Atopic dermatitis model non-human animal and use thereof

ABSTRACT

An atopic dermatitis model non-human animal, containing a gene mutation in which a complex containing dedicator of cytokinesis 8 (DOCK8) protein, mammalian STE20-like kinase 1 (MST1) protein, and endothelial PAS domain protein 1 (EPAS1) protein is not formed in CD4 +  T cells.

TECHNICAL FIELD

The present invention relates to an atopic dermatitis model non-humananimal and use thereof. More specifically, the present invention relatesto an atopic dermatitis model non-human animal, a potential drug targetfor an atopic dermatitis treatment, a method for screening a therapeuticagent for atopic dermatitis, a therapeutic agent for atopic dermatitis,and a non-human animal for developing an atopic dermatitis model animal.

Priority is claimed on Japanese Patent Application No. 2016-034510,filed on Feb. 25, 2016, the content of which is incorporated herein byreference.

BACKGROUND ART

Atopic dermatitis is a chronic inflammatory skin disease, and patientsthereof are increasing all over the world. Atopic dermatitis ischaracterized by the formation of erythematous exudative lesionsaccompanied by inflammatory infiltrates composed mainly of CD4-positive(CD4⁺) T cells.

Itching and scratching behavior play important roles in the onset anddeterioration of skin inflammation, and therefore identification of apruritogen is important for effective treatment strategies for atopicdermatitis. In addition, at present, interleukin (IL)-31 is consideredto be a major pruritogen.

IL-31 is produced from activated helper type 2 (T_(II)2) CD4⁺ T cells.Intradermal injection of IL-31 to mice and transgenic miceoverexpressing IL-31 exhibit increased scratching behavior and causesevere dermatitis. In addition, it is known that in patients with atopicdermatitis, cutaneous lymphocyte antigen (CLA)-positive skin homing CD4⁺T cells produce IL-31, and the IL-31 concentration in serum correlateswith the severity of the disease. Furthermore, a result of a phase IIclinical trial revealed that the itching behavior is suppressed when ananti-IL-31 receptor antibody is administered to the patients with atopicdermatitis.

Therefore, IL-31 is a cytokine derived from T cells and has a closerelationship with itching in atopic dermatitis.

Incidentally, a hyper IgE syndrome is a complex primary immunodeficiencydisorder characterized by atopic dermatitis accompanied with high serumIgE levels and susceptibility to infections with extracellular bacteria.It has recently been reported that the autosomal recessive type of hyperIgE syndrome is caused mainly by mutations in the dedicator ofcytokinesis 8 (DOCK8) gene (refer to, for example, NPL 1).

It is known that DOCK8 is an evolutionarily conserved guanine nucleotideexchange factor (GEF) for activating Cdc42. The present inventorsgenerated DOCK8-knockout (DOCK8^(−/−)) mice and have revealed that DOCK8is essential for interstitial migration of dendritic cells (refer to,for example, NPL 2).

CITATION LIST Patent Literature

-   [NPL 1] Zhang Q, et al., Combined Immunodeficiency Associated with    DOCK8 Mutations, N. Engl. J. Med., 361 (21), 2046-2055, 2009.-   [NPL 2] Harada Y, et al., DOCK8 is a Cdc42 activator critical for    interstitial dendritic cell migration during immune responses,    Blood, 119 (19), 4451-4461, 2012.

SUMMARY OF INVENTION Technical Problem

As seen above, IL-31 is the major pruritogen in atopic dermatitis, but amechanism regulating IL-31 production by CD4⁺ T cells remains unknown.An object of the present invention is to clarify the mechanismregulating IL-31 production and to provide an atopic dermatitis modelnon-human animal.

Solution to Problem

The present invention includes the following aspects.

[1] An atopic dermatitis model non-human animal, having a gene mutationin which a complex containing dedicator of cytokinesis 8 (DOCK8)protein, mammalian STE20-like kinase 1 (MST1) protein, and endothelialPAS domain protein 1 (EPAS1) protein is not formed in CD4⁺ T cells.

[2] The atopic dermatitis model non-human animal according to [1], inwhich the gene mutation is knockout or knockdown of DOCK8 gene or MST1gene.

[3] The atopic dermatitis model non-human animal according to [1] or[2], in which a rearranged T cell receptor (TCR) is expressed.

[4] The atopic dermatitis model non-human animal according to any one of[1] to [3], further having a genotype of DOCK8^(−/−) TCR Tg orMST1^(−/−) TCR Tg (herein, TCR

Tg represents a rearranged TCR transgene).

[5] The atopic dermatitis model non-human animal according to [3] or[4], in which the TCR is AND.

[6] An atopic dermatitis model cell, having a gene mutation in which acomplex containing DOCK8 protein, MST1 protein, and EPAS1 protein is notformed.

[7] The cell according to [6], in which the gene mutation is knockout orknockdown of DOCK8 gene or MST1 gene.

[8] A method for screening a therapeutic agent for atopic dermatitis,including: quantitatively determining a degree of scratching behavior ofthe non-human animal according to any one of [1] to [5] underadministration of a test substance; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe quantitatively determined degree of the scratching behavior isdecreased when compared to a degree of the scratching behavior of thenon-human animal under non-administration of the test substance.

[9] A method for screening a therapeutic agent for atopic dermatitis,including: stimulating TCR of CD4⁺ T cells from a patient with atopicdermatitis, DOCK8^(−/−) CD4⁺ T cells, or MST1^(−/−) CD4⁺ T cells in thepresence of a test substance to quantitatively determine an expressionlevel of interleukin (IL)-31 by the CD4⁺ T cells; and determining thatthe test substance is the therapeutic agent for atopic dermatitis in acase where the expression level of the IL-31 is decreased when comparedto an expression level of the IL-31 in a case where the TCR of the CD4⁺T cells is stimulated in the absence of the test substance.

[10] A method for screening a therapeutic agent for atopic dermatitis,including: expressing EPAS1 gene in T cells in the presence of a testsubstance to quantitatively determine an expression level of IL-31; anddetermining that the test substance is the therapeutic agent for atopicdermatitis in a case where the expression level is decreased whencompared to an expression level of the IL-31 in a case where the EPAS1gene is expressed in the cells in the absence of the test substance.

[11] A method for screening a therapeutic agent for atopic dermatitis,including: expressing EPAS1 gene in cells into which a reporterconstruct in which a reporter gene is linked downstream of an IL-31promoter is introduced in the presence of a test substance toquantitatively determine an expression level of the reporter gene; anddetermining that the test substance is the therapeutic agent for atopicdermatitis in a case where the expression level of the reporter gene isdecreased when compared to an expression level of the reporter gene in acase where the EPAS1 gene is expressed in the cells in the absence ofthe test substance.

[12] A method for screening a therapeutic agent for atopic dermatitis,including: stimulating TCR of DOCK8^(−/−) T cells or MST1^(−/−) T cellsin the presence of a test substance to quantitatively determine anexpression level of IL-31; and determining that the test substance isthe therapeutic agent for atopic dermatitis in a case where theexpression level is decreased when compared to an expression level ofthe IL-31 in a case where the TCR of the cells is stimulated in theabsence of the test substance.

[13] A method for screening a therapeutic agent for atopic dermatitis,including: stimulating TCR of DOCK8^(−/−) T cells or MST1^(−/−) T cells,into which a reporter construct in which a reporter gene is linkeddownstream of an IL-31 promoter is introduced in the presence of a testsubstance to quantitatively determine an expression level of thereporter gene; and determining that the test substance is thetherapeutic agent for atopic dermatitis in a case where the expressionlevel of the reporter gene is decreased when compared to an expressionlevel of the reporter gene in a case where the TCR of the cells isstimulated in the absence of the test substance.

[14] A method for screening a therapeutic agent for atopic dermatitis,including: measuring a binding force between EPAS1 protein and DOCK8protein in the presence of a test substance; and determining that thetest substance is the therapeutic agent for atopic dermatitis in a casewhere the binding force is increased when compared to the binding forcebetween the EPAS1 protein and the DOCK8 protein in the absence of thetest substance.

[15] A method for screening a therapeutic agent for atopic dermatitis,including: measuring a binding force between EPAS1 protein and MST1protein in the presence of a test substance; and determining that thetest substance is the therapeutic agent for atopic dermatitis in a casewhere the binding force is increased when compared to the binding forcebetween the EPAS1 protein and the MST1 protein in the absence of thetest substance.

[16] A method for screening a therapeutic agent for atopic dermatitis,including: measuring a binding force between DOCK8 protein and MST1protein in the presence of a test substance; and determining that thetest substance is the therapeutic agent for atopic dermatitis in a casewhere the binding force is increased when compared to the binding forcebetween the DOCK8 protein and the MST1 protein in the absence of thetest substance.

[17] A method for screening a therapeutic agent for atopic dermatitis,including: measuring a binding force between EPAS1 protein and SP1protein in the presence of a test substance; and determining that thetest substance is the therapeutic agent for atopic dermatitis in a casewhere the binding force is decreased when compared to the binding forcebetween the EPAS1 protein and the SP1 protein in the absence of the testsubstance.

[18] A method for screening a therapeutic agent for atopic dermatitis,including: expressing EPAS1 gene in DOCK8^(−/−) cells or MST1^(−/−)cells in the presence of a test substance to quantitatively determinethe abundance of nuclear EPAS1 protein; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe abundance of the nuclear EPAS1 protein is decreased when compared toabundance of the nuclear EPAS1 protein in a case where the EPAS1 gene isexpressed in the cells in the absence of the test substance.

[19] A method for screening a therapeutic agent for atopic dermatitis,including: quantitatively determining an expression level of EPAS1 incells in the presence of a test substance; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe expression level is decreased when compared to an expression levelof the EPAS1 in the cells in the absence of the test substance.

[20] EPAS1 as a potential drug target of a therapeutic agent for atopicdermatitis. [21] A therapeutic agent for atopic dermatitis whichcontains an EPAS1 inhibitor as an active ingredient.

[22] A non-human animal for generating an atopic dermatitis modelanimal, having a genotype of DOCK8^(+/−) TCR Tg, DOCK8^(+/−),DOCK8^(−/−), MST1^(+/−) TCR Tg, MST1^(+/−), MST1^(−/−), or TCR Tg(herein, TCR Tg represents a rearranged TCR transgene).

[23] The non-human animal for generating an atopic dermatitis modelanimal according to [22], in which the TCR is AND.

[24] A method for generating an atopic dermatitis model non-humananimal, including: crossing a non-human animal having a genotype ofDOCK8^(+/−)TCR Tg with a non-human animal having a genotype ofDOCK8^(+/−) TCR Tg, DOCK8^(+/−), or DOCK8^(−/−), in which a non-humananimal having a genotype of DOCK8^(−/−) TCR Tg appearing in offspring isthe atopic dermatitis model non-human animal (herein, TCR Tg representsa rearranged TCR transgene).

[25] A method for generating an atopic dermatitis model non-humananimal, including: crossing a non-human animal having a genotype ofMST1^(+/−) TCR Tg with a non-human animal having a genotype ofMST1^(+/−) TCR Tg, MST1^(+/−), or MST1^(−/−), in which a non-humananimal having a genotype of MST1^(−/−) TCR Tg appearing in offspring isthe atopic dermatitis model non-human animal (herein, TCR Tg representsa rearranged TCR transgene).

[26] A method for generating an atopic dermatitis model non-human animalaccording to [24] or [25], in which the TCR is AND.

Advantageous Effects of Invention

According to the present invention, it is possible to clarify themechanism regulating IL-31 production and provide an atopic dermatitismodel non-human animal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of Experimental Example 1. (a) is a representativephotograph of 18-week-old DOCK8^(+/−) AND Tg mice. (b) is arepresentative photograph of 18-week-old DOCK8^(−/−) AND Tg nice. (c) isa graph showing the incidence of atopic dermatitis of male mice (n=20)having a genotype of DOCK8^(−/−) AND Tg. (d) is a graph showing theincidence of the atopic dermatitis of female mice (n=15) having thegenotype of DOCK8^(−/−) AND Tg.

FIG. 2 is a graph showing quantitative measurements of scratchingbehaviors of DOCK8+/− AND Tg mice and DOCK8−/− AND Tg mice inExperimental Example 2.

FIG. 3 shows results of Experimental Example 3. (a) and (b) arephotographs of the results of hematoxylin and eosin staining of the skinof the 18-week-old DOCK8^(+/−) AND Tg mice (a) and the DOCK8^(−/−) ANDTg littermate mice (b). (c) is a graph showing total number ofinflammatory cells per 0.25 mm² in the skin of the DOCK8^(+/−) AND Tgmice and the DOCK8^(−/−) AND Tg mice.

(a) and (b) of FIG. 4 are fluorescence microscopy photographs of theresults in which the skin of the DOCK8^(+/−) AND Tg mice (a) and theDOCK8^(−/−) AND Tg mice (b) is stained with an anti-CD3 antibody and ananti-CD4 antibody as described in Experimental Example 3. (c) and (d)are fluorescence microscopy photographs of the results in which the skinof the DOCK8^(+/−) AND Tg mice (c) and the DOCK8^(−/−) AND Tg mice (d)is stained with the anti-CD3 antibody, an anti-CD8 antibody, and ananti-B220 antibody in Experimental Example 3.

(a) and (b) of FIG. 5 are graphs of the results of measuringconcentrations of IgE (a) and IgG2b (b) in sera of the DOCK8^(+/−) ANDTg mice and the DOCK8^(−/−) AND Tg mice in Experimental Example 4.

FIG. 6 is a graph of IL-31 concentration in the sera of the DOCK8^(+/−)AND Tg mice and the DOCK8^(−/−) AND Tg mice in Experimental Example 5.

FIG. 7 is the results of flow cytometry analysis in Experimental Example6.

FIG. 8 is the results of flow cytometry analysis in Experimental Example6.

FIG. 9 is a graph of the results of examining antigen-specificproliferation of CD4⁺ T cells in Experimental Example 7.

FIG. 10 shows results of Experimental Example 8. (a) is a graph showingthe expression of the IL-31 gene, (b) is a graph showing the expressionof the IL-4 gene, and (c) is a graph showing the expression of the IL-2gene.

FIG. 11 is a graph showing the expression level of the IL-31 gene insecondary stimulated CD4⁺ T cells in Experimental Example 8.

FIG. 12 is a graph of the results of IL-31 protein concentrationmeasured by ELISA for the culture supernatant of the secondarystimulated CD4⁺ T cells in Experimental Example 9.

FIG. 13 is a graph showing the expression level of IL-31 gene in CD4⁺ Tcells primary stimulated in the presence of anti-IL-4 antibody inExperimental Example 10.

FIG. 14 shows the results of the flow cytometry analysis in ExperimentalExample 11.

FIG. 15 is a graph showing antigen-specific proliferation of the CD4⁺ Tcells in Experimental Example 12.

FIG. 16 is a graph showing the expression level of the IL-31 gene in thesecondary stimulated CD4⁺ T cells in Experimental Example 13.

FIG. 17 is a graph of the results of IL-31 protein concentrationmeasured by ELISA for the culture supernatant of the secondarystimulated CD4⁺ T cells in Experimental Example 14.

FIG. 18 is a graph showing IL-31 concentration in sera of DOCK8^(+/−)OTII Tg mice and DOCK8^(−/−) OTII Tg mice in Experimental Example 15.

(a) and (b) of FIG. 19 are photographs of the results of hematoxylin andeosin staining of the skin of 18-week-old DOCK8^(+/−) OTII Tg mice (a)and DOCK8^(−/−) OTII Tg littermate mice (b) in Experimental Example 16.(c) is the results of the total number of inflammatory cells per 0.25mm² in the skin of the DOCK8^(+/−) OTII Tg mice (n=4) and theDOCK8^(−/−) OTII Tg mice (n=4) in Experimental Example 16.

FIG. 20 is a graph showing the expression level of IL-31 gene in theCD4⁺ T cells in Experimental Example 17.

(a) of FIG. 21 is a graph showing the expression level of the IL-31 genein Experimental Example 18. (b) is a graph of the results of RT-PCR inExperimental Example 18.

FIG. 22 is a graph showing the expression level of the IL-31 gene inExperimental Example 19.

FIG. 23 is a representative photograph of CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice and control littermate mice inExperimental Example 20.

FIG. 24 is a graph showing quantitative measurements of scratchingbehaviors of the CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice and thecontrol littermate mice in Experimental Example 20.

(a) of FIG. 25 shows photographs of the results of hematoxylin and eosinstaining of the skin of the CD4-Cre⁺ EPAS1^(lox/lox) DOCK8^(−/−) AND Tgmice and the control littermate mice in Experimental Example 20. (b) isa graph of the total number of inflammatory cells per 0.25 mm² in theskin of the CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice and thecontrol littermate mice in Experimental Example 20.

FIG. 26 is a graph showing the concentration of the IL-31 concentrationin the sera of the CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice andthe control littermate mice in Experimental Example 20.

(a) of FIG. 27 is a schematic diagram of EPAS1 mutants used inExperimental Example 21. (b) is a graph showing the activity of IL-31promoter in the presence of the EPAS1 mutants in Experimental Example21.

(a) of FIG. 28 is a graph showing the activity of the IL-31 promoter inExperimental Example 22. (b) are photographs of Western blotting showingthe effect of knockdown of ARNT gene in Experimental Example 22. (c) arephotographs of Western blotting showing the effect of knockdown of SP1gene in Experimental Example 22.

FIG. 29 is a graph showing the activity of IL-31 promoter inExperimental Example 23.

FIG. 30 is a graph showing the activity of IL-31 promoter inExperimental Example 24.

(a) and (b) of FIG. 31 are photographs showing the results of EMSA inExperimental Example 25.

FIG. 32 is a graph of the results of ChIP assay in Experimental Example26.

(a) of FIG. 33 shows fluorescence microscopy photographs of EPAS1^(−/−)mouse embryonic fibroblasts (MEF) stained with anti-EPAS1 antibody inExperimental Example 27. (b) shows fluorescence microscopy photographsof a wild-type MEF stained with anti-EPAS1 antibody in ExperimentalExample 27.

(a) and (b) of FIG. 34 are fluorescence microscopy photographs of theresults of the immunofluorescence staining in Experimental Example 27.(c) is a graph showing the proportion of cells with nuclear localizationof EPAS1 in Experimental Example 27.

(a) of FIG. 35 is a fluorescence microscopy photograph of the results ofthe immunofluorescence staining in Experimental Example 28. (b) is agraph showing the proportion of the cells with nuclear localization ofthe EPAS1 in Experimental Example 28.

(a) and (b) of FIG. 36 are photographs of the results ofimmunoprecipitation in Experimental Example 29.

FIG. 37 shows photographs of Western blotting showing the effect ofknockdown of MST1 gene in Experimental Example 30.

(a) to (c) of FIG. 38 are fluorescence microscopy photographs of theresults of the immunofluorescence staining in Experimental Example 30.(d) is a graph showing the proportion of the cells with nuclearlocalization of EPAS1 in Experimental Example 30.

(a) of FIG. 39 is a graph showing the expression level of the IL-31 genein Experimental Example 31. (b) is a graph of the results of the RT-PCRin Experimental Example 31.

FIG. 40 shows photographs of the results of the Western blotting inExperimental Example 32.

FIG. 41 is a graph showing the concentration of the IL-31 in serum of anatopic dermatitis patient and a healthy subject (control) inExperimental Example 33.

(a) of FIG. 42 is a graph showing the expression level of the IL-31 genein Experimental Example 34. (b) is a graph showing the expression levelof IL-2 gene in Experimental Example 34.

FIG. 43 shows photographs showing the validity of the anti-EPAS1antibody by the Western blotting in Experimental Example 35.

FIG. 44 shows photographs showing the effect of EPAS1 inhibitors onEPAS1 expression by the Western blotting in Experimental Example 35.

(a) of FIG. 45 is a graph showing the effect of FM19G11 on theexpression level of IL-31 gene in Experimental Example 36. (b) is agraph showing the effect of FM19G11 on the expression level of IL-2 genein Experimental Example 36. (c) is a graph showing the effect of HIFVIIon the expression level of the IL-31 gene in Experimental Example 36.

(a) and (b) of FIG. 46 are photographs of the results of theimmunoprecipitation in Experimental Example 37.

FIG. 47 is a graph of the results of quantitative assessment ofdermatitis of DOCK8-conditional knockout mice and control mice inExperimental Example 38.

FIG. 48 is a graph showing quantitative measurements of scratchingbehaviors of CAG-OVA mice following adoptive transfer of either CD4⁺ Tcells from DOCK8^(+/−) OTII Tg mice or DOCK8^(−/−) OTII Tg mice inExperimental Example 39.

DESCRIPTION OF EMBODIMENTS

[Atopic Dermatitis Model Non-Human Animal]

In one embodiment, the present invention provides an atopic dermatitismodel non-human animal which has a gene mutation so as not to form acomplex comprising DOCK8 protein, MST1 protein, and EPAS1 protein inCD4⁺ T cells.

The non-human animal is not particularly limited, and examples thereofinclude a mouse, a rat, a rabbit, a pig, a cow, a monkey, and the like.The atopic dermatitis model non-human animal of the present embodimenthas a gene mutation through which a complex comprising DOCK8 protein,MST1 protein, and EPAS1 protein is not formed in the CD4⁺ T cells.

In the present specification, the atopic dermatitis model non-humananimal means a non-human animal which is a model for atopic dermatitis.The model for atopic dermatitis means an experimental system capable ofreproducing at least a part of onset mechanisms of the atopic dermatitisor symptoms of atopic dermatitis. For example, CD4⁺ T cells derived fromthe atopic dermatitis model non-human animal of the present embodimentproduce large amounts of IL-31 on TCR stimulation, and therefore it canbe used as a model for atopic dermatitis. In addition, after CD4⁺ Tcells derived from the atopic dermatitis model non-human animal of thepresent embodiment are stimulated and transferred to another non-humananimal (recipient), the atopic dermatitis develops in the recipient, andthus such recipient of the non-human animal can be used as a model foratopic dermatitis. In the related art, the mechanism of how CD4⁺ T cellsproduce large amounts of the IL-31 on the TCR stimulation has not beenknown. Therefore, the atopic dermatitis model non-human animal of thepresent embodiment can be effectively used for development of atherapeutic agent and a therapeutic method for atopic dermatitis.

In the atopic dermatitis model non-human animal of the presentembodiment, a rearranged T cell receptor (TCR) may be expressed. Therearranged TCR is not particularly limited as long as it is TCR thatrecognizes a specific antigen, and examples thereof include AND, OTII,and the like. The AND and the OTII will be described later. Theabove-described TCR may have self-reactivity. For example, as will bedescribed in examples later, the AND is considered to be a TCR havingself-reactivity.

In one embodiment, the present invention provides an atopic dermatitismodel non-human animal which has the gene mutation in which the complexcomprising DOCK8 protein, MST1 protein, and EPAS1 protein is not formedin the CD4⁺ T cells, and in which the TCR having self-reactivity isexpressed. As will be described in the examples later, the atopicdermatitis spontaneously develops in the atopic dermatitis modelnon-human animal of the present embodiment.

In NC/Nga mice, which have been used as an atopic dermatitis model micein the related art, the atopic dermatitis is caused due to parasiticmites. However, in the NC/Nga mice, the atopic dermatitis does notdevelop under specific-pathogen-free (SPF) environments. In addition,there is a case where the NC/Nga mice do not show scratching behavioreven when the dermatitis occurs, and there is a case where an individualexcessively exhibiting dermatitis does not show an itching reaction. Asabove, the NC/Nga mice have different symptoms from human atopicdermatitis patients.

With respect to the above description, the atopic dermatitisspontaneously develops in the atopic dermatitis model non-human animalof the present embodiment even under SPF environments. In addition, theatopic dermatitis model non-human animal of the present embodiment isstable in symptoms of the dermatitis and the itching reaction, and showssymptoms extremely similar to those of human atopic dermatitis patients,in that the itching reaction becomes strong, and the concentration ofIL-31 in serum increases, in accordance with progression of the symptomsof the dermatitis.

Therefore, the atopic dermatitis model non-human animal of the presentembodiment is extremely useful for clarifying the pathogenesis of humanatopic dermatitis, developing a therapeutic drug and a therapeuticmethod, and the like.

As will be described in the examples later, the present inventors haveclarified the mechanism of regulating IL-31 expression that is apruritogen of atopic dermatitis. That is, in CD4⁺ T cells of a wildtype, the complex containing the DOCK8 protein, the MST1 protein, andEPAS1 protein is formed, and therefore the EPAS1 protein is localizedwithin the cytoplasm. With respect to the above description, if theabove complex cannot be formed, the EPAS1 protein is translocated intothe nucleus and induces IL-31 expression in association with SP1protein. Association between EPAS1 and the SP1 is important forinduction of transcription of the IL-31.

In addition, it is known that the EPAS1 forms a complex with an arylhydrocarbon receptor nuclear translocator (also called ARNT, HIF-1β),and activates transcription of a target gene in response toenvironmental stress such as hypoxia, for example. In addition, it hasbeen reported that the ARNT is involved in neurogenesis and control oflumen formation of trachea/salivary gland, and it is known that the ARNTis an important factor in development. Accordingly, there is a concernthat drugs affecting the functions of ARNT may have severe side effects.

However, as will be described in the examples later, the presentinventors have revealed that the EPAS1 functions independently of theARNT and induces IL-31 production. That is, it has been revealed thatthe EPAS1 activates the IL-31 promoter independently of the ARNT, but incollaboration with the SP1.

The above description means that, in a case where the EPAS1 is atherapeutic target, there is less concern that causes severe sideeffects. That is, the EPAS1 is suitable as a therapeutic target foratopic dermatitis.

Therefore, in the one embodiment, the present invention provides theEPAS1 as a potential drug target of the therapeutic target for atopicdermatitis.

Note that RefSeq ID of human DOCK8 protein is NP_982272, and RefSeq IDof mouse DOCK8 protein is NP_083061. In addition, RefSeq ID of humanMST1 protein is NP_006273, and RefSeq ID of mouse MST1 protein isNP_067395. In addition, RefSeq ID of human EPAS1 protein is NP_001421,and RefSeq ID of mouse EPAS1 protein is NP_034267.

In the atopic dermatitis model non-human animal of the presentembodiment, the gene mutation in which the tri-molecular complexcomprising the DOCK8 protein, the MST1 protein, and the EPAS1 protein isunformable is not particularly limited as long as it is a mutation bywhich the EPAS1 protein is localized in the nucleus, and examples of themutation may be knockout of DOCK8 gene or MST1 gene, may be a knockdownof the DOCK8 gene or the MST1 gene, or may be a gene mutation in whichan N terminal side of the DOCK8 protein is deleted.

As will be described in the examples later, the present inventors haverevealed that EPAS1 is localized in the nucleus of the cells when DOCK8is knocked out, 527 amino acids of the N terminal side of the DOCK8protein are deleted, and the knockdown of the MST1 gene, and the like,and therefore the IL-31 gene expression increases following TCRstimulation.

The DOCK8 gene or the MST1 gene may be knocked out in all organs or maybe knocked out only with a cell population containing the CD4⁺ T cellsby conditional knockout or the like.

In the atopic dermatitis model non-human animal of the presentembodiment, in addition to the gene mutation as described above, CD4⁺ Tcells may express a (rearranged) TCR transgene (TCR Tg) whichreorganizes a specific antigen. That is, the atopic dermatitis modelnon-human animal of the present embodiment may have a genotype ofDOCK8^(−/−) TCR Tg or MST1^(−/−) TCR Tg. Herein, TCR Tg represents arearranged TCR transgene.

As will be described in the examples later, the present inventors haverevealed that in CD4⁺ T cells where nuclear transportation of the EPAS1is likely to occur, large amounts of IL-31 are induced following TCRstimulation. As a result, the concentration of the IL-31 in serumincreases, and therefore atopic dermatitis develops.

Examples of the rearranged TCR include the AND. It is known that the ANDis the TCR that recognizes a peptide (SEQ ID NO: 1) consisting of 88thto 103rd amino acids of Moth cytochrome c (MCC), which forms a complexwith MHC class II I-E^(K) molecule, but the AND recognizes I-A^(b)molecules in the thymus to differentiate and mature (refer to, forexample, Kaye J. et al., Selective development of CD4⁺ T cells intransgenic mice expressing a class II MHC-restricted antigen receptor,Nature 341, 746-749, 1989).

The atopic dermatitis model non-human animal of the present embodimentmay have a genotype of DOCK8^(−/−) AND Tg or MST1^(−/−) AND Tg.

In the non-human animal having the genotype of DOCK8^(−/−) AND Tg, DOCK8gene is deleted, and therefore the CD4⁺ T cells cannot form the complexcontaining the DOCK8 protein, the MST1 protein, and EPAS1 protein. Inaddition, these CD4⁺ T cells produce large amounts of the IL-31, andthus the atopic dermatitis spontaneously develops in this non-humananimal.

Furthermore, in the non-human animal having the genotype of MST1^(−/−)AND Tg, the MST1 gene is deleted, and therefore the CD4⁺ T cells cannotform the complex containing the DOCK8 protein, the MST1 protein, andEPAS1 protein. In addition, these CD4⁺ T cells produce large amounts ofthe IL-31, and thus the atopic dermatitis spontaneously develops in thisnon-human animal.

In the present specification, the term “AND Tg” means that AND TCR isexpressed, and may be AND^(Tg/−) or may be AND^(Tg/Tg). However, in thenon-human animal having the genotype of AND^(Tg/Tg), there is a casewhere an unintentional gene is deleted through the integrated positionof the AND gene on genome, and therefore AND^(Tg/−) is preferable.

The non-human animal of the present embodiment may be in a form of anindividual or in a form of a fertilized egg or embryo. In theabove-described individual, atopic dermatitis spontaneously develops bybreeding. In addition, the fertilized egg or embryo is transplanted intothe uterus of the non-human animal so as to be grown into an individual,and thus can be used.

[Atopic Dermatitis Model Cell]

In one embodiment, the present invention provides an atopic dermatitismodel cell which has a gene mutation in which the tri-molecular complexcomprising DOCK8 protein, MST1 protein, and EPAS1 protein is not formed.

The cells of the present embodiment may be cells harvested from theabove-described atopic dermatitis model non-human animal, or may be thecultured cells in which the DOCK8 gene or the MST1 gene is knocked outor knocked down. The cultured cells are not particularly limited, andfor example, human cells can be used. The cells may be, for example,cells of a hematopoietic cell type, and may be, for example, cells of anon-hematopoietic cell type such as fibroblasts.

The cells of the present embodiment can be used for, for example, amethod of screening a therapeutic agent for atopic dermatitis, whichwill be described later.

[Non-Human Animal for Generating Atopic Dermatitis Model Animal andMethod for Generating Atopic Dermatitis Model Non-Human Animal]

In one embodiment, the present invention provides a non-human animal forgenerating an atopic dermatitis model animal, which contains a genotypeof DOCK8^(+/−) TCR Tg, DOCK8^(+/−), DOCK8^(−/−), MST1^(+/−) TCR Tg,MST1^(+/−), MST1^(−/−), or TCR Tg (herein, TCR Tg represents arearranged TCR transgene). The rearranged TCR is not particularlylimited as long as it is TCR that recognizes a specific antigen, andexamples thereof include AND, OTII, and the like.

In a case where the above-described TCR is the AND, the non-human animalfor generating an atopic dermatitis model animal of the presentembodiment has a genotype of DOCK8^(+/−) AND Tg, DOCK8^(+/−),DOCK8^(−/−), MST1^(+/−) AND Tg, MST1^(+/−), MST1^(−/−), or AND Tg.

In the non-human animal having the genotype of DOCK8^(+/−) AND Tg,DOCK8^(+/−), DOCK8^(−/−), MST1^(+/−) AND Tg, MST1^(−/−), or AND Tg,atopic dermatitis does not spontaneously develop. However, by crossingthese non-human animals, an atopic dermatitis model non-human animal inwhich atopic dermatitis spontaneously develops appears in offspringthereof. Accordingly, it can be said that these non-human animals are anon-human animal for generating an atopic dermatitis model animal.

For example, in the non-human animal having the genotype of DOCK8^(+/−)AND Tg, atopic dermatitis does not develop. However, by crossing thisnon-human animal with, for example, a non-human animal having thegenotype of DOCK8^(+/−) AND Tg, DOCK8^(+/−), DOCK8^(−/−), and the like,a non-human animal having the genotype of DOCK8^(−/−) AND Tg appears inoffspring thereof. This non-human animal is an atopic dermatitis modelnon-human animal in which atopic dermatitis spontaneously develops.

Similarly, in the non-human animal having the genotype of MST1^(+/−) ANDTg, atopic dermatitis does not develops. However, by crossing thisnon-human animal with, for example, a non-human animal having thegenotype of MST1^(+/−) AND Tg, MST1^(+/−), MST1^(−/−), and the like, thenon-human animal having the genotype of MST1^(−/−) AND Tg appears inoffspring thereof. This non-human animal is an atopic dermatitis modelnon-human animal in which atopic dermatitis spontaneously develops.

That is, in the one embodiment, the present invention provides a methodfor generating an atopic dermatitis model non-human animal, whichincludes crossing a non-human animal having a genotype of DOCK8^(+/−)TCR Tg with a non-human animal having a genotype of DOCK8^(+/−) TCR Tg,DOCK8^(+/−), or DOCK8^(−/−). Herein, TCR Tg represents a rearranged TCRtransgene. The rearranged TCR is not particularly limited as long as itis TCR that recognizes a specific antigen, and examples thereof includeAND, OTII, and the like.

Among the offspring of the non-human animal obtained by the generationmethod of the present embodiment, for example, a non-human animal havingthe genotype of DOCK8^(−/−) AND Tg is an atopic dermatitis modelnon-human animal in which the atopic dermatitis spontaneously develops.

In addition, among the offspring of the non-human animal obtained by thegeneration method of the present embodiment, for example, in thenon-human animal having the genotype of DOCK8^(−/−) or DOCK8^(−/−) OTIITg, the atopic dermatitis does not develops. However, the CD4⁺ T cellsderived from such non-human animal produce large amounts of IL-31following TCR stimulation, and therefore such non-human animal is usedas a model for atopic dermatitis. In addition, after CD4⁺ T cellsderived from the non-human animal having the genotype of DOCK8^(−/−),DOCK8^(−/−) OTII Tg, or the like are stimulated and transferred intoanother non-human animal (recipient), and the like, atopic dermatitisspontaneously develops in the non-human animal (recipient), andtherefore the non-human animal can be used as a model for atopicdermatitis.

In addition, in one embodiment, the present invention provides a methodfor generating a atopic dermatitis model non-human animal, whichincludes crossing a non-human animal having a genotype of MST1^(+/−) TCRTg with a non-human animal having a genotype of MST1^(+/−) TCR Tg,MST1^(+/−), or MST1^(−/−). Herein, TCR Tg represents a rearranged TCRtransgene. The rearranged TCR is not particularly limited as long as itis TCR that recognizes a specific antigen, and examples thereof includeAND, OTII, and the like.

Among the offspring of the non-human animal obtained by the productionmethod of the present embodiment, for example, the non-human animalhaving the genotype of MST1^(−/−) AND Tg is an atopic dermatitis modelnon-human animal in which atopic dermatitis spontaneously develops.

In addition, among the offspring of the non-human animal obtained by thegenerating method of the present embodiment, for example, in thenon-human animal having the genotype of MST1^(−/−) or MST1^(−/−) OTIITg, atopic dermatitis does not develop spontaneously. However, the CD4⁺T cells derived from the non-human animal produce large amounts of IL-31following TCR stimulation, and therefore the non-human animal is used asa model for atopic dermatitis. In addition, after CD4⁺ T cells derivedfrom the non-human animal having the genotype of MST1^(−/−), MST1^(−/−)OTII Tg, or the like are stimulated and transferred into anothernon-human animal (recipient), and the like, the atopic dermatitis iscaused in the non-human animal (recipient), and therefore the non-humananimal can be used as a model for atopic dermatitis.

[Method for Screening Therapeutic Agent for Atopic Dermatitis]

First Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofquantitative measurements of scratching behavior of the above-describedatopic dermatitis model non-human animal under administration of a testsubstance; and determination that the test substance is the therapeuticagent for atopic dermatitis in a case where the quantitativelydetermined degree of the scratching behavior is decreased, compared tothe degree of scratching behavior in the absence of the test substanceadministration.

The scratching behavior can not be evaluated at a cell level, and thusneeds to be evaluated at an individual level. As described above, theabove-described atopic dermatitis model non-human animal shows extremelysimilar symptoms as those of the human atopic dermatitis. For thisreason, by the method for screening of the present embodiment, it ispossible to efficiently screen a therapeutic agent effective for thehuman atopic dermatitis.

As the test substance, for example, a compound library or the like canbe used, and the same applies for a screening method to be describedlater. In addition, therapeutic agents for atopic dermatitis, which areobtained by any one of screening methods described later, or a candidatesubstance thereof may be used as the test substance.

A method for administering the test substance is not particularlylimited, and for example, the test substance may be administered orally,may be administered to the blood by injection or the like, or may beapplied to the skin.

A method for quantitative measurements of scratching behavior is notparticularly limited. Examples of the method include a measurement of afrequency of the scratching behavior. The frequency of the scratchingbehavior can be measured by, for example, playing recordings obtained byvideotaping the non-human animal for a predetermined time frame,counting the number of the scratching behaviors, and the like.

In a case where the degree of the scratching behavior is decreased byadministration of the test substance, it can be determined that thecorresponding test substance is a therapeutic agent for atopicdermatitis. As a mechanism of such a test substance, suppression ofIL-31 production, blocking a signaling pathway downstream of IL-31receptor, and the like are considered.

Second Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofstimulating the TCR of the CD4⁺ T cells from a patient with atopicdermatitis, DOCK8^(−/−) CD4⁺ T cells or MST1^(−/−) CD4⁺ T cells in thepresence of the test substance to quantitatively compare the expressionlevel of the IL-31 by the CD4⁺ T cells; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe expression level of the IL-31 is decreased compared to that in theabsence of the test substance.

The expression level of the IL-31 may be quantitatively determined at atranscription level by, for example, quantitative real-time PCR or thelike, or may be quantitatively measured at a protein level by, forexample, an ELISA method or the like.

As will be described in the examples later, if the TCR of the CD4⁺ Tcells from the patient with the atopic dermatitis, DOCK8^(−/−) CD4⁺ Tcells or MST1^(−/−) CD4⁺ T cells are stimulated, the expression level ofthe IL-31 is increased. Therefore, the substance that suppresses suchincrease in the expression level of the IL-31 is a candidate therapeuticagent for atopic dermatitis. In other words, the therapeutic agent foratopic dermatitis can also be called an IL-31 expression inhibitor.

The DOCK8^(−/−) CD4⁺ T cells and the MST1^(−/−) CD4⁺ T cells may beharvested from the above-described atopic dermatitis model non-humananimal overproducing the IL-31, or may be cells in which the DOCK8 geneor the MST1 gene is knocked out or knocked down in an established T cellline or the like. In addition, in the DOCK8^(−/−) CD4⁺ T cells and theMST1^(−/−) CD4⁺ T cells, a rearranged TCR may be expressed.

In addition, the TCR stimulation may be performed by the antigenspecific to its TCR, or may be performed with an anti-TCR antibody.Furthermore, as will be described in the examples later, by performingthe TCR stimulation twice, the expression level of the IL-31 can beremarkably increased.

Third Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofexpressing the EPAS1 gene in T cells in the presence of the testsubstance to quantitatively compare the expression level of IL-31; anddetermining that the test substance is the therapeutic agent for atopicdermatitis in a case where the expression level is decreased compared tothe level in the absence of the test substance.

As will be described in the examples later, if the EPAS1 gene isoverexpressed in the CD4⁺ T cells, the expression level of IL-31 isincreased. Therefore, the substance that suppresses the increase in theexpression level of IL-31 is a candidate therapeutic agent for atopicdermatitis. In other words, the therapeutic agent for atopic dermatitiscan also be called an IL-31 expression inhibitor.

The T cells may be primary T cells, may be an established T cell line,or may be a thymocyte cell line.

Fourth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofexpressing EPAS1 gene in cells having a reporter construct composed ofIL-31 promoter region in the presence of the test substance to seequantitatively measure the expression level of the reporter gene; anddetermining that the test substance is the therapeutic agent for atopicdermatitis in a case where the expression level of the reporter gene isdecreased compared to the level of an expression level of the reportergene in the absence of the test substance.

As will be described in the examples later, the present inventors haveclarified that IL-31 promoter activation can be induced in the presenceof the EPAS1 by a reporter assay. As such, the substance that suppressesthe IL-31 promoter activation is a candidate therapeutic agent foratopic dermatitis. In other words, the therapeutic agent for atopicdermatitis can also be called an IL-31 expression inhibitor.

Examples of the IL-31 promoter include a promoter of human IL-31 shownin SEQ ID NO: 2, a promoter of mouse IL-31 shown in SEQ ID NO: 3, or thelike. Examples of the reporter gene include luciferase gene. The EPAS1gene may be, for example, human EPAS1 gene. A method for controllingEPAS1 gene expression is not particularly limited, and may be performedby, for example, introducing an expression vector of the EPAS1 gene intocells, transiently overexpressing the gene, and the like. Alternatively,the expression of the EPAS1 gene may be regulated by using atetracycline expression induction system or the like capable ofregulating ON and OFF of target gene expression by addition oftetracycline or doxycycline, for example. Alternatively, endogenouslyexpressed EPAS1 gene may be used.

The cells are not particularly limited, and for example, human cells canbe used. The cells may be, for example, cells of hematopoietic celltype, or may be, for example, cells of the non-hematopoietic cell typesuch as fibroblasts.

Fifth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofstimulating the TCR of DOCK8^(−/−) T cells or MST1^(−/−) T cells in thepresence of the test substance to quantitatively compare the expressionlevel of IL-31; and determining that the test substance is thetherapeutic agent for atopic dermatitis in a case where the expressionlevel of IL-31 is decreased compared to the level in the absence of thetest substance.

As will be described in the examples later, when the TCR of theDOCK8^(−/−) CD4+ T cells or MST1^(−/−) CD4⁺ T cells is stimulated, theexpression level of IL-31 is increased. Therefore, the substance thatsuppresses the increase in the expression level of IL-31 is a candidatetherapeutic agent for atopic dermatitis. In other words, the therapeuticagent for atopic dermatitis can also be called an IL-31 expressioninhibitor.

The DOCK8^(−/−) T cells and the MST1^(−/−) T cells may be cellsharvested from the above-described atopic dermatitis model non-humananimal, or may be cells in which the DOCK8 gene or the MST1 gene isknocked out or knocked down in the primary T cells, an established Tcell line, a thymocyte cell line, or the like.

The TCR stimulation may be performed by the antigen specific to the TCR,or may be performed with anti-TCR antibody.

Sixth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofstimulating the TCR of the DOCK8^(−/−) T cells or MST1^(−/−) T cellsexpressing the reporter construct composed of the IL-31 promoter regionin the presence of test substance to quantitatively measure theexpression level of the reporter gene; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe expression level of the reporter gene is decreased compared to anexpression level of the reporter gene in the absence of the testsubstance.

As will be described in the examples later, when the TCR of theDOCK8^(−/−) CD4⁺ T cells or MST1^(−/−) CD4⁺ T cells is stimulated, theexpression level of IL-31 is increased. Therefore, the substance thatsuppresses the increase in the expression level of IL-31 is a candidatetherapeutic agent for atopic dermatitis. In other words, the therapeuticagent for atopic dermatitis can also be called an IL-31 expressioninhibitor.

Examples of the IL-31 promoter include the promoter of the human IL-31shown in SEQ ID NO: 2, the promoter of the mouse IL-31 shown in SEQ IDNO: 3, or the like. Examples of the reporter gene include the luciferasegene.

The DOCK8^(−/−) T cells and the MST1^(−/−) T cells may be harvested fromthe above-described atopic dermatitis model non-human animal, or may becells in which the DOCK8 gene or the MST1 gene is knocked out or knockeddown in the primary T cells, an established T cell line, a thymocytecell line, or the like.

In addition, the TCR stimulation may be performed by antigen specific tothe TCR, or may be performed with the anti-TCR antibody.

Seventh Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofmeasuring a binding force between EPAS1 protein and DOCK8 protein in thepresence of the test substance; and determining that the test substanceis the therapeutic agent for atopic dermatitis in a case where thebinding force is increased compared to that in the absence of the testsubstance.

As will be described in the examples later, in the CD4⁺ T cells of thewild type, the complex containing the DOCK8 protein, the MST1 protein,and EPAS1 protein is formed, and therefore EPAS1 is localized within thecytoplasm. In contrast, if the above complex cannot be formed, EPAS1 istranslocated into the nucleus and induces IL-31 gene expression incollaboration with SP1.

Accordingly, the substance that increases the binding force betweenEPAS1 protein and the DOCK8 protein can be used as a candidatetherapeutic agent for atopic dermatitis. The binding force between EPAS1protein and the DOCK8 protein can be measured by, for example, the ELISAmethod, immunoprecipitation, BIACORE (registered trademark), or thelike. Herein, in the ELISA method or the immunoprecipitation, it can besaid that as a binding amount between EPAS1 protein and the DOCK8protein becomes high, the binding force between them becomes high. Inaddition, in the measurement by the BIACORE (registered trademark), itcan be said that, for example, as a K_(D) value becomes small, thebinding force becomes high.

The binding force between EPAS1 protein and the DOCK8 protein may bemeasured using a cell sample in which the test substance is added to amedium. Alternatively, the binding force may be measured in a test tubeusing purified EPAS1 protein and purified DOCK8 protein, in the presenceof a test substance.

Eighth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofmeasuring a binding force between EPAS1 and the MST1 protein in thepresence of the test substance; and determining that the test substanceis the therapeutic agent for atopic dermatitis in a case where thebinding force is increased compared to that in the absence of the testsubstance.

As will be described in the examples later, in the CD4⁺ T cells of thewild type, the complex containing the DOCK8 protein, the MST1 protein,and EPAS1 is formed, and therefore EPAS1 is localized within thecytoplasm. If the above complex cannot be formed, EPAS1 is transportedinto the nucleus and induces IL-31 gene expression in collaboration withSP1.

Accordingly, the substance that increases the binding force betweenEPAS1 and the MST1 protein can be used as a candidate therapeutic agentfor atopic dermatitis. The binding force between EPAS1 and the MST1protein can be measured in the same manner as that of the binding forcebetween EPAS1 and the DOCK8 protein, which was described above.

Ninth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofmeasuring a binding force between the DOCK8 protein and the MST1 proteinin the presence of the test substance; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe binding force is increased compared to that in the absence of thetest substance.

As will be described in the examples later, in the CD4⁺ T cells of thewild type, the complex containing DOCK8 protein, MST1 protein, and EPAS1protein is formed, and therefore EPAS1 protein is localized within thecytoplasm. If the above complex cannot be formed, EPAS1 is transportedinto the nucleus and induces IL-31 gene expression in collaboration withSP1.

Accordingly, it can be said that the substance that increases thebinding force between DOCK8 protein and MST1 protein also has an actionof allowing the EPAS1 to localize within the cytoplasm. Therefore, thesubstance that increases the binding force between DOCK8 protein andMST1 protein can be used as a candidate therapeutic agent for atopicdermatitis. The binding force between DOCK8 protein and MST1 protein canbe measured in the same manner as that of the binding force betweenEPAS1 protein and DOCK8 protein, which was described above.

Tenth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofmeasuring a binding force between EPAS1 protein and SP1 protein in thepresence of the test substance; and determining that the test substanceis the therapeutic agent for atopic dermatitis in a case where thebinding force is decreased compared to that in the absence of the testsubstance.

As will be described in the examples later, if EPAS1 is transported intothe nucleus, EPAS1 induces the IL-31 gene expression in collaborationwith the SP1.

Accordingly, the substance that decreases the binding force betweenEPAS1 protein and SP1 protein can be used as a candidate therapeuticagent for atopic dermatitis. The binding force between EPAS1 and SP1 canbe measured in the same manner as that of the binding force betweenEPAS1 and the DOCK8 protein, which was described above.

Eleventh Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofexpressing the EPAS1 gene in DOCK8^(−/−) cells or MST1^(−/−) cells inthe presence of the test substance to quantitatively measure theabundance of nuclear EPAS1 protein; and determining that the testsubstance is the therapeutic agent for atopic dermatitis in a case wherethe abundance of the nuclear protein is decreased compared to that inthe absence of the test substance.

As will be described in the examples later, the present inventors haveclarified that, in normal cells, EPAS1 is localized in the cytoplasmunder normal conditions, and EPAS1 is translocated into the nucleusunder hypoxia. In addition, it has been clarified that, in DOCK8^(−/−)cells or MST1^(−/−) cells, EPAS1 is likely to translocate into thenucleus even under the normal conditions.

Accordingly, in the DOCK8^(−/−) cells or MST1^(−/−) cells under thenormal conditions, the substance that decreases the abundance of nuclearEPAS1 protein is a candidate therapeutic agent for atopic dermatitis.Herein, the term “under normal conditions” means a condition under whichan oxygen concentration is normal (about 20% (v/v)).

The DOCK8^(−/−) cells or MST1^(−/−) cells may be harvested from theabove-described atopic dermatitis model non-human animal, or may becells in which the DOCK8 gene or the MST1 gene is knocked out or knockeddown in cultured cells. The cultured cells are not particularly limited,and for example, human cells can be used. The cells may be, for example,cells of the hematopoietic cell type, and may be, for example, cells ofthe non-hematopoietic cell type such as fibroblasts.

Twelfth Embodiment

In one embodiment, the present invention provides a method for screeningthe therapeutic agent for atopic dermatitis, which includes a step ofquantitatively determining the expression level of the EPAS1 in thecells in the presence of the test substance; and determining that thetest substance is the therapeutic agent for atopic dermatitis in a casewhere the expression level is decreased compared to that in the absenceof the test substance.

As will be described in the examples later, EPAS1 induces IL-31 geneexpression in collaboration with SP1 protein. Accordingly, the substancethat decreases the expression level of EPAS1 is a candidate therapeuticagent for atopic dermatitis.

The expression level of the EPAS1 may be quantitatively determined atthe transcriptional level by, for example, quantitative real-time PCR orthe like, or may be quantitatively determined at the protein level by,for example, the Western blotting method or the like.

In addition, the cells may be harvested from the above-described atopicdermatitis model non-human animal, may be cells in which the DOCK8 geneor the MST1 gene is knocked out or knocked down, or may be cells havinga wild-type genotype. The cells may be, for example, human cells. Inaddition, the cells may be, for example, cells of the hematopoietic celltype, and may be, for example, cells of the non-hematopoietic cell typesuch as fibroblasts.

[Potential Drug Target of Therapeutic Agent for Atopic Dermatitis]

In one embodiment, the present invention provides the EPAS1 as apotential drug target of the therapeutic agent for atopic dermatitis.The EPAS1 may be a gene that encodes EPAS1, or may be EPAS1.

As will be described in the examples later, the present inventors haveclarified that the EPAS1 has new properties such as (1) controllingIL-31 gene expression, and (2) functioning independently of the ARNTprotein involved in the neurogenesis and control of lumen formation ofthe trachea/salivary gland. Accordingly, the EPAS1 is suitable for useas the potential drug target of the therapeutic agent for atopicdermatitis.

[Therapeutic Agent for Atopic Dermatitis]

In one embodiment, the present invention provides a therapeutic agentfor atopic dermatitis which contains an EPAS1 inhibitor as an activeingredient.

The EPAS1 inhibitor means a substance that inhibits the functions of theEPAS1. As will be described in the examples later, EPAS1 induces IL-31gene expression in collaboration with SP1 protein. Accordingly, theEPAS1 inhibitor is a candidate therapeutic agent for atopic dermatitis.

Examples of the EPAS1 inhibitor include an expression-inhibitorysubstance of the EPAS1, a substance that decreases the IL-31 geneexpression by EPAS1, and the like. Examples of the substance thatdecreases IL-31 gene expression by EPAS1 include a substance thatspecifically binds to EPAS1 to inhibit the EPAS1-SP1 interaction, andsubstance that specifically binds to SP1 to inhibit the EPAS1-SP1interaction, and the like.

(Expression-Inhibitory Substance)

Examples of the expression-inhibitory substance include siRNA, shRNA, aribozymes, an antisense nucleic acid, a low-molecular-weight compound,or the like. By administering these expression-inhibitory substances toa living body, EPAS1 expression can be inhibited. As a result, IL-31gene expression can be suppressed, and therefore it is possible to treatthe atopic dermatitis. Examples of the above-describedlow-molecular-weight compound include FM19611(3-[(2,4-dinitrobenzoyl)amino]-benzoic acid2-(4-methylphenyl)-2-oxoethyl ester, [2-oxo-2-(p-tolyl)ethyl]3-[(2,4-dinitrobenzoyl)amino]benzoate, refer to, for example,Moreno-Manzano V., et al., FM19G11, a new hypoxia-inducible factor (HIF)modulator, affects stern cell differentiation status., J. Biol. Chem.,285 (2), 1333-1342, 2010).

Small interfering RNA (siRNA) is double-stranded RNA small molecules of21 to 23 base pairs used for gene silencing via RNA interference. ThesiRNA introduced into a cell binds to RNA-induced silencing complex(RISC). This complex binds to and cleaves mRNA that has a complementarysequence with the siRNA. Therefore, gene expression is suppressed in asequence-specific manner.

The siRNA can be prepared by respectively synthesizing sense andantisense oligonucleotides with an automated DNA/RNA synthesizer,denaturing the oligonucleotides in, for example, an appropriateannealing buffer at 90° C. to 95° C. for about 1 minute, and thenannealing of the oligonucleotide at 30° C. to 70° C. for about 1 to 8hours.

Short hairpin RNA (shRNA) is a hairpin type RNA sequence used for genesilencing via RNA interference. The shRNA may be introduced into a cellby a vector and expressed with a U6 promoter or an H1 promoter, or maybe prepared by synthesizing an oligonucleotide having an shRNA sequencewith the automated DNA/RNA synthesizer, and self-annealing of theoligonucleotide in the same method as that of the siRNA. A hairpinstructure of the shRNA introduced into the cell is cleaved into thesiRNA and binds to the RNA-induced silencing complex (RISC). Thiscomplex binds to and cleaves the mRNA that has the complementarysequence with the siRNA. Therefore, gene expression is suppressed in asequence-specific manner.

Ribozyme is RNA having catalytic activity. The ribozyme has variousactivities, but studies on the ribozyme as an enzyme that cleaves RNAmake it possible to design the ribozyme aimed at site-specific cleavageof RNA. The ribozyme may be of a size of 400 nucleotides or more, suchas a group I intron type, or M1 RNA contained in RNase P, or may haveabout 40 nucleotides, which is called a hammerhead type, a hairpin type,or the like.

The antisense nucleic acid is a nucleic acid complementary to a targetsequence. The antisense nucleic acid can suppress the expression of atarget gene by inhibition of transcription initiation by triplexformation, suppression of transcription by hybridization with a sitewhere an opened-loop structure is locally formed by RNA polymerase,inhibition of transcription by hybridization with RNA still in theprocess of synthesis, suppression of splicing by hybridization with ajunction of intron and exon, suppression of splicing by hybridizationwith a spliceosome-formation site, suppression of migration from nucleusto cytoplasm by hybridization with mRNA, suppression of splicing byhybridization with a capping site and a poly(A) addition site,inhibition of translation initiation by hybridization with a translationinitiation factor binding site, inhibition of translation byhybridization with a ribosome binding site around a start codon,inhibition of elongation of a peptide chain by hybridization with a mRNAtranslation region and a polysome binding site, suppression of geneexpression by hybridization with a site of interaction between a nucleicacid and a protein, or the like.

The siRNA, the shRNA, the ribozyme, and the antisense nucleic acid mayinclude various chemical modifications in order to improve stability andactivity thereof. For example, phosphate residues may be substitutedwith chemically modified phosphate residues such as phosphorothioate(PS), methylphosphonate, and phosphorodithionate, in order to preventdegradation by a hydrolase such as a nuclease. In addition, at least apart thereof may be constituted by a nucleic acid analog such as peptidenucleic acid (PNA).

(Specific Binding Substance) The substance that specifically binds toEPAS1 can inhibit its association with, for example, SP1 protein throughthe binding to EPAS1. As a result, IL-31 expression can be suppressed,and therefore it is possible to treat the atopic dermatitis. Similarly,the substance that specifically binds to SP1 can inhibit its associationwith, for example, EPAS1 through the binding to SP1. As a result, IL-31gene expression can be suppressed, and therefore it is possible to treatthe atopic dermatitis.

Examples of the specific binding substance include an antibody, anantibody fragment, an aptamer, a low-molecular-weight compound, or thelike. The antibody can be produced by, for example, immunizing an animalsuch as a mouse with an antigen. Alternatively, the antibody can beproduced by screening antibody libraries such as phage libraries, or thelike.

Examples of the antibody fragment include Fv, Fab, scFv, or the like.The above-described antibody or antibody fragment may be polyclonal ormonoclonal. In addition, the above-described antibody or antibodyfragment may be an antibody or antibody fragment to which a compoundsuch as polyethylene glycol is bound. By being bound with thepolyethylene glycol, it becomes possible to increase blood stability,for example.

The aptamer is a substance having a specific binding ability withrespect to a labeled substance. Examples of the aptamer include anucleic acid aptamer, a peptide aptamer, or the like. The nucleic acidaptamer having the specific binding ability with respect to EPAS1 can beselected by, for example, the systematic evolution of ligands byexponential enrichment (SELEX) method or the like. In addition, thepeptide aptamer having the specific binding ability with respect toEPAS1 can be selected by, for example, a yeast two-hybrid method, or thelike.

In addition to the above-described examples, the specific bindingsubstance may be obtained by screening a compound library or the like,with a binding property to a target substance as a reference forexample.

(Substance Decreasing IL-31 Gene Expression by EPAS1 Protein) It can besaid that a substance that decreases EPAS1-mediated IL-31 geneexpression is a candidate therapeutic agent for atopic dermatitis. Sucha substance can be screened from the compound library or the like by anyof the screening methods described above, for example.

(Dosage Form and Dosage) The above-described therapeutic agent foratopic dermatitis may itself be administered to a living body, or may beadministered to a living body in a form of being formulated as apharmaceutical composition mixed with a pharmaceutically acceptablecarrier.

The pharmaceutical composition may be formulated into a dosage form tobe used orally, such as tablets, capsules, elixirs, or microcapsules,and may be formulated into a dosage form to be used parenterally, suchas injections, ointments, or patches.

Examples of the pharmaceutically acceptable carrier include solventssuch as sterilized water and physiological salt solution; binders suchas gelatin, corn starch, tragacanth gum, and gum arabic; excipients suchas crystalline cellulose; swelling agents such as corn starch, gelatin,and alginic acid, or the like.

The pharmaceutical composition may contain an additive. Examples of theadditive include lubricants such as magnesium stearate; sweeteners suchas sucrose, lactose, and saccharin; flavoring agents such as peppermintand Gaultheria adenothrix oil; stabilizers such as benzyl alcohol andphenol; buffers such as phosphate, and sodium acetate; solubilizers suchas benzyl benzoate and benzyl alcohol; antioxidants; preservatives;surfactants; emulsifiers, or the like.

The pharmaceutical composition can be formulated by appropriatelycombining the above-described carriers and additives, and mixing thecarriers and additives in unit dosage form required for generallyaccepted pharmaceutical practice.

In a case where the pharmaceutical composition is the injection,examples of a solvent for the injection include isotonic solutionscontaining adjuvants such as physiological salt solution, glucose,D-sorbitol, D-mannose, D-mannitol, and sodium chloride. The solvent forthe injection may contain alcohols such as ethanol; polyalcohols such aspropylene glycol and polyethylene glycol; nonionic surfactants such aspolysorbate 80 (trademark) and HCO-50, or the like.

Administration of the therapeutic agent for atopic dermatitis to thepatient can be carried out intranasally, transbronchially,intramuscularly, transdermally, or orally by methods known to thoseskilled in the art, in addition to intraarterial injection, intravenousinjection, subcutaneous injection, or the like.

The dosage of the therapeutic agent for atopic dermatitis variesdepending on symptoms, but in the case of oral administration, thedosage is generally, for example, 0.1 to 100 mg, for example, 1 to 50mg, for example, 1 to 20 mg, or the like per day for an adult (bodyweight of 60 kg).

In the case of parenteral administration, the single dosage variesdepending on a subject to be administered, a target organ, a symptom,and an administration method. For example, in the form of the injection,the dosage of about, for example, 0.01 to 30 mg, for example, 0.1 to 20mg, or, for example, 0.1 to 10 mg per day is administered for an adult(body weight of 60 kg) by intravenous injection or local injection.

OTHER EMBODIMENTS

In one embodiment, the present invention provides a method for treatingatopic dermatitis, which includes administering an effective dose of theEPAS1 inhibitor to patients who need to be treated or to animals withthe atopic dermatitis.

In such embodiment, the present invention provides a method forinhibiting IL-31 gene expression, which includes administering aneffective dose of the EPAS1 inhibitor to patients who need to be treatedor to animals with the atopic dermatitis.

In such embodiment, the present invention provides the EPAS1 inhibitorfor treating the atopic dermatitis.

In such embodiment, the present invention provides use of the EPAS1inhibitor for producing the therapeutic agent for atopic dermatitis.

EXAMPLES

Next, the present invention will be described in more detail by showingexperimental examples, but the present invention is not limited to thefollowing experimental examples.

[Materials and Methods] (Mice) DOCK8^(−/−) mice were developed inadvance (refer to NPL 2). Hetero mice (DOCK8^(+/−)) were backcrossedonto a C57BL/6 background for more than nine generations. DOCK8^(+/−)mice were crossed with AND TCR Tg mice to obtain DOCK8^(+/−) AND Tg miceor DOCK8^(−/−) AND Tg mice. EPAS1^(lox/lox) mice were obtained from theJackson Laboratory and crossed with CD4-Cre Tg mice to obtain CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice. Mice were bred underspecific-pathogen-free (SPF) environments in the animal facility ofKyushu University. Age- and sex-matched littermates were used ascontrols. All animal experiments were approved by the committee ofEthics of Animal Experiments, Kyushu University, and carried outaccording to guidelines. Mice were selected randomly and assigned toeach experimental group according to genotype. Experimenters whoperformed the experiments were blinded to mouse genotypes.

(Measurement of Scratching Behavior) Mice were put into an acrylic cageof 11×14×20 cm for at least 1 hour for acclimation, and then thebehaviors of the mice were videotaped. The video was played fordetermination of a total number of the scratching behaviors per 2-hourperiod.

(Histology and Immunofluorescence Staining) Skin tissues were fixed in4% (w/v) paraformaldehyde and embedded in paraffin blocks. Sectionshaving a thickness of 3 μm were stained with hematoxylin and eosin, andobserved with a bright-field microscope. For immunofluorescenceanalyses, tissues were embedded in OCT compound (Sakura Finetech) andfrozen in liquid nitrogen. Frozen sections having a thickness of 10 μmwere fixed in 4% (w/v) paraformaldehyde for 30 minutes and blocked with10% goat serum for 1 hour. The sections were then stained withphycoerythrin (PE)-conjugated anti-mouse CD3 antibody (model “17A2”,Biolegend), fluorescein isothiocyanate (FITC)-conjugated anti-mouseCD45R/B220 antibody (model “RA3-6B2”, BD Biosciences), FITC-conjugatedanti-mouse CD8a antibody (model “53-6.7”, BD Biosciences), orbiotinylated anti-mouse CD4 antibody (model “H129.19”, BD Biosciences),followed by incubation with Alexa Fluor 488-conjugated streptavidin(Thermo Fisher Scientific). For EPAS1 staining, mouse embryonicfibroblasts (MEF) (3×10⁵ cells/mL) were cultured on the poly-L-lysinecoated glass-bottom dishes (Matsunami) in 1% 02 environment for 30hours, fixed with 4% (w/v) paraformaldehyde for 30 minutes, andpermeabilized with 0.2% Triton X-100 for 30 minutes. After being blockedwith 10% goat serum for 1 hour at room temperature, cells were thenstained with 4′,6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories)and rabbit anti-EPAS1 antibody (NOVUS Biologicals), followed byincubation with Alexa Fluor 488-conjugated donkey anti-rabbit IgGantibody (Fab fragment, Jackson ImmunoReseach). All images were capturedwith a laser scanning confocal microscope (Carl Zeiss).

(ELISA)

IL-31 concentrations in serum samples and cell culture supernatants weremeasured with ELISA kits (R&D Systems for human samples and USCN formouse samples), according to the manufacturer's instructions.Measurement of the concentrations of serum IgE and IgG2b was carried outas follows. First, the serum samples were serially diluted and placed in96-well plates coated with goat anti-mouse IgE antibody or goatanti-mouse Ig (IgM+IgG+IgA, H+L) antibody. After 2-hour incubation, thewells were washed with phosphate-buffered saline (PBS) and incubatedwith alkaline phosphatase-conjugated rat anti-mouse IgE antibody or goatanti-mouse IgG2b antibody (Southern Biotech).

(Flow Cytometry)

The following antibodies and reagents were used. FITC-conjugatedanti-mouse CD45R/B220 antibody (model “RA3-6B2”), FITC-conjugatedanti-mouse CD4 antibody (model “RM4-5”), biotinylated anti-mouse CD4antibody (model “RM4-5”), PE-conjugated anti-mouse CD8a antibody (model“53-6.7”), PE-conjugated anti-mouse CD44 antibody (model “1M7”),FITC-conjugated anti-mouse CD62L antibody (model “MEL-14”),FITC-conjugated anti-mouse Vα11 antibody (model “RR8-1”),FITC-conjugated anti-mouse Vα2 antibody (model “B20.1”), biotinylatedanti-mouse Vβ3 antibody (model “KJ25”), biotinylated anti-mouse Vβ5antibody (model “MR9-4”), and allophycocyanin (APC)- orPerCP-cyanine5.5-conjugated streptavidin antibody (all of which werefrom BD Biosciences), and biotinylated anti-mouse CD90.2/Thy1.2 (model“30-H12”, eBioscience). Before staining with the antibodies, cells wereincubated for 10 minutes on ice with anti-Fcγ III/III receptor antibody(model “2.4G2”, BD Biosciences) to block Fc receptors. Flow cytometricanalyses were carried out using FACS Calibur (trade name, BDBiosciences).

(Preparation and Culture of Cells)

Mouse CD4⁺ T cells were isolated from the spleen and peripheral lymphnodes by magnetic sorting with Dynabeads mouse CD4, followed bytreatment with DETACHaBEAD mouse CD4 (Life Technologies), and suspendedin RPMI 1640 medium (Wako) containing 10% heat-inactivated fetal calfserum (FCS), 50 μM 2-mercaptoethanol (Nacalai Tesque), 2 mM L-glutamine(Life Technologies), 100 U/mL 1 penicillin, 100 μg/mL streptomycin, 1 mMsodium pyruvate (Life Technologies), and MEM non-essential amino acids(Life Technologies). For performing primary stimulation, CD4⁺ T cells(3×10⁵ cells/well) were cultured in a 24-well plate with Tcell-depleted, irradiated spleen cells (5×10⁶ cells/well) in thepresence of MCC88-103 peptide (SEQ ID NO: 1, 3 μg/mL) or OVA323-339peptide (SEQ ID NO: 4, 1 μg/mL).

For performing secondary stimulation, CD4⁺ T cells recovered from theculture were re-stimulated with plate-bound anti-CD3ε antibody (model“145-2C11”, eBioscience, 1 μg/mL), and with anti-CD28 antibody (model“37.51, BD Biosciences, 1 μg/mL) for some cases.

T cell proliferation assays were carried out by cultivating CD4⁺ T cells(5×10⁴ cells/well) with T cell-depleted, irradiated spleen cells (1×10⁶cells/well) in the presence or absence of various concentrations of thepredetermined peptide for 66 hours. [³H]-thymidine (0.037 MBq) was addedduring the final 18 hours of the culture, and the incorporatedradioactivity was measured with a liquid scintillation counter.

Human peripheral blood samples were collected from atopic dermatitispatients and healthy volunteers in compliance with Institutional ReviewBoard protocols of the facility. Human peripheral blood mononuclearcells (PBMC) were separated by Percoll (GE Healthcare) gradientcentrifugation. The CD4⁺ T cells were isolated from PBMCs by magneticsorting by using Dynabeads human CD4 followed by treatment withDETACHaBEAD human CD4 (Life Technologies).

After being suspended in complete RPMI 1640 medium, CD4⁺ T cells (3×10⁵cells/well) were stimulated in a 24-well plate coated with anti-humanCD3E antibody (model “Hit3a”, Tonbo Biosciences) for 6 hours. In someexperiments, the CD4⁺ T cells were treated with EPAS1 inhibitors,FM19G11 or HIFVII at 30 μM (both from Calbiochem).

These experiments were approved by the Ethics committee of KyushuUniversity Hospital, and a written informed consent was obtained fromall patients and the healthy volunteers.

(RT-PCR and Quantitative Real-Time PCR)

Total RNA was extracted using ISOGEN (trade name, Nippon Gene). Aftertreatment with RNase-free DNase I (Life Technologies), RNA samples werereverse-transcribed with oligo (dT) primers (Life Technologies) andSuperScript III reverse transcriptase (Life Technologies) foramplification by PCR.

Sequence numbers of base sequences of primers used for RTPCR of eachgene are listed in Table 1. Note that, in the present specification,GAPDH is an abbreviation for glyceraldehyde triphosphate dehydrogenase.

TABLE 1 Gene Sense primer Antisense primer Mouse GAPDH SEQ ID NO: 5 SEQID NO: 6 Mouse IL-31 SEQ ID NO: 7 SEQ ID NO: 8 Mouse EPAS1 SEQ ID NO: 9SEQ ID NO: 10 Mouse MST1 SEQ ID NO: 11 SEQ ID NO: 12

Real-time PCR was performed using ABI PRISM 7,000 Sequence DetectionSystem (trade name, Applied Biosystems) and SYBR Green PCR Master Mix(Applied Biosystems).

Sequence numbers of base sequences of primers used for real-time PCR arelisted in Table 2. Note that, in the present specification, HPRT is anabbreviation of hypoxanthine-guanine phosphoribosyltransferase.

TABLE 2 Gene Sense primer Antisense primer Human GAPDH SEQ ID NO: 13 SEQID NO: 14 Human IL-31 SEQ ID NO: 15 SEQ ID NO: 16 Human IL-2 SEQ ID NO:17 SEQ ID NO: 18 Mouse HPRT SEQ ID NO: 19 SEQ ID NO: 20 Mouse IL-31 SEQID NO: 21 SEQ ID NO: 22 Mouse IL-4 SEQ ID NO: 23 SEQ ID NO: 24 MouseIL-2 SEQ ID NO: 25 SEQ ID NO: 26

The expression levels of human and mouse target genes were normalizedbased on expression levels of GAPDH and HPRT. Sequence detectionsoftware attached to real-time PCR equipment was used for analyses.

(Microarray Analysis)

Total RNA was extracted using ISOGEN (trade name, Nippon Gene). cRNA wasamplified and labelled using a commercially available kit (model “LowInput Quick Amp Labeling Kit”, Agilent Technologies).

The cRNA was then hybridized to a microarray (model “44K 60-meroligomicroarray (Whole Mouse Genome oligo DNA Microarray Kit Ver 2.0”,Agilent Technologies). The hybridized microarray slides were scannedusing a scanner manufactured by Agilent Technologies. The relativehybridization intensities and background hybridization values werecalculated using software (model “Feature Extraction Software version9.5.1.1”, Agilent Technologies). Raw signal intensities and flags foreach probe were calculated based on the hybridization intensities andspot information, according to the procedures recommended by AgilentTechnologies. To identify up- or down-regulated genes in experimentalsamples, the present inventors calculated Z-scores and ratios based onthe normalized signal intensities of each probe (up-regulated genes,Z-score>2.0 and ratio>1.5-fold; down-regulated genes, Z-score<−2.0 andratio<0.66-fold).

(Knockdown of Target Genes by siRNAs)

To knock down EPAS1 gene or MST1 gene expression in DOCK8^(−/−) orDOCK8^(+/−) AND Tg CD4⁺ T cells, a commercially available kit (tradename, model “Accell siRNA SMART pool”, model “E-040635-00-0005” (forEPAS1) or model “E-059385-00-0005” (for MST1) (Dharmacon) was used.

Transfection was performed according to the manufacturer's instructionsusing oligonucleotide (trade name “Accell Red Non-targeting siRNA”,model “D-001960-01-05”, Dharmacon) as a control.

Briefly, DOCK8^(−/−) or DOCK8^(+/−) AND Tg CD4⁺ T cells (3×10⁵cells/well) were cultured with T cell-depleted, irradiated spleen cells(5×10⁶ cells/well) and MCC88-103 peptide (SEQ ID NO: 1, 3 μg/mL) inmedium (model “Accell siRNA Delivery Media”, Dharmacon) supplementedwith 2.5% FCS. The siRNA or the control oligonucleotide was then addedto the culture at the final concentration of 1 μM. After 4 days of theculture, viable CD4⁺ T cells were recovered and re-stimulated withplate-bound anti-CD3ε antibody (model “145-2C11”, 1 μg/mL) and anti-CD28antibody (model “37.51”, 1 μg/mL) for 3 hours. The knockdown efficacywas checked by RT-PCR.

In a case of knockdown of SP1 gene and ARNT gene expression in MEFs, thesiRNAs (trade name “On-Target plus SMART pool”, model “L-040633-02-0005”(for SP1) or “L-040639-01-0005” (for ARNT), Dharmacon) and irrelevantoligonucleotide (trade name “BLOCK-iT Alexa Fluor Red”, LifeTechnologies) were used.

For transfection, first, 400 μL of the siRNA solution (150 nM) inserum-free DMEM medium (Wako) was mixed with 5 μL DharmaFecttransfection reagent, and left to stand for 20 minutes at roomtemperature. This solution was added drop-wise to MEFs (3×10⁵cells/well) suspended in 1.6 mL DMEM medium containing 10% FCS. Then,cells were incubated for 24 hours at 37° C. before transienttransfection for luciferase reporter assays. The knockdown efficacy waschecked by Western blot analyses.

(Plasmids and Transfection)

To generate mouse IL-31 reporter plasmid (pGL4.10-IL-31), a promoterregion of the mouse IL-31 gene from position 1,367 to 1 was amplified byPCR and subcloned into pGL4.10 [luc2] vector (Promega). Deletions andmutations in the IL-31 promoter region were created by PCR. Theseplasmids were transfected into MEFs with Lipofectamine 2,000 reagent(Life Technologies) for luciferase reporter assays.

The pcDNA vector (Invitrogen) was used to create expression vectorsencoding FLAG-tagged human and mouse EPAS1 (pcDNA-EPAS1) or its deletionmutants, HA-tagged mouse DOCK8 (pcDNA-DOCK8), FLAG-tagged or V5-taggedmouse MST1 (pcDNA-MST1), and HA-tagged human ARNT (pcDNA-ARNT).

These expression constructs were transfected into HEK-293T cells withpolyethyleminine for immunoprecipitation.

The pBJ vector (pBJ-neo) encoding neomycin was used to create expressionvectors encoding WT (wild type) DOCK8 and its mutants, mutants (ΔN)lacking the N-terminal 527 amino acid residues of DOCK8, and mutants(ΔDHR2) lacking amino acid residues from 1,535 to 2,100 of DOCK8.

The retroviral vector pMX was used to generate pMX-EPAS1-IRES-GFP (greenfluorescent protein) plasmid.

This plasmid DNA was transfected into Platinum-E packaging cells (COSMOBIO) with FuGENE 6 transfection reagent (Promega). The cell culturesupernatants were harvested 48 hours after transfection, supplementedwith polybrene (5 μg/mL) and IL-2 (5 ng/mL), and were used to infect theCD4⁺ T cells.

After centrifugation at 2000 rpm for 1 hour, plates were incubated for 8hours at 32° C. and for 16 hours at 37° C. Two additional retroviralinfections were performed at daily intervals, and the GFP-positive CD4⁺T cells were sorted by FACSAria (BD Biosciences) for RNA extraction 30hours after the third transfection.

To generate the retroviral vector pSUPER retro-puro sh MST1, adouble-stranded DNA fragment consisting of oligonucleotides (SEQ ID NOs:27 and 28) corresponding to specific regions of MST1 was ligated to thepSUPER retro-puro vector (OligoEngine) at the Bgl II and Hind III sites.

MEFs were retrovirally transduced with pSUPER retro-puro sh MST1 asdescribed above.

(Luciferase Reporter Assays)

MEFs were co-transfected with pRL-SV40-Renilla luciferase plasmid (0.1μg, Promega) and pGL4.10-IL-31 (2 μg), and pcDNA-EPAS1 or its mutants (2μg).

For transfection, these plasmid DNAs were mixed with Lipofectamine 2,000transfection reagent (5 μL) in 500 μL of the Opti-DMEM medium (LifeTechnologies), and left to stand for 20 minutes at room temperature, andthe mixture was added drop-wise to MEFs (3×10⁵ cells/well) cultured in1.5 mL of DMEM medium containing 1% FCS. 6 hours after transfection,cells were suspended in DMEM containing 10% FCS and incubated foradditional 24 hours. In some experiments, pGL4.10-IL-31-derived mutantswere used.

A total amount of plasmid DNA was equalized by the control vector.Luciferase activity was measured with a commercially available kit(model “Dual-Luciferase Reporter Assay System”, Promega) according tothe manufacture's protocols.

(Immunoblotting and Immunoprecipitation)

Cells were lysed in 20 mM Tris-HCl buffer (pH 7.5) containing 1% TritonX-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1 mM Na₃VO₄, and complete protease inhibitors(Roche).

After centrifugation, the supernatants were mixed with an equal volumeof 2× sample buffer (125 mM Tris-HCl, 0.01% bromophenacyl bromide, 4%SDS, 20% glycerol, and 200 μM dithiothreitol). Samples were boiled for 5minutes and analyzed by immunoblotting.

The following antibodies were used. Rabbit anti-EPAS1 antibody (NovusBiologicals), rabbit anti-SP1 antibody (Millipore), rabbit anti-ARNTantibody (Cell Signaling), rabbit anti-MST1 antibody (Cell Signaling),goat anti-bactin antibody (model “1-19”, Santa Cruz) rat anti-HAantibody (model “3F10”, Roche), anti-FLAG antibody (MBL) and anti-V5antibody (Life Technologies).

Polyclonal antibody against DOCK8 was produced by immunizing rabbitswith KLH-coupled synthetic peptide corresponding to the C-terminalsequence of human and mouse DOCK8 (2081 to 2100, SEQ ID NO: 29). In someexperiments, immunoblotting was performed by immunoprecipitation ofHEK-293T cell lysates with the appropriate antibodies. Signals weremeasured using software (software name “ImageJ”,http://imagej.nih.gov/ij/) and normalized to β-actin.

(EMSA)

Nuclear extracts were prepared from MEFs using a standard method.Briefly, cells were rinsed with PBS, resuspended in buffer A (10 mMHEPES-K⁺, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mMPMSF) and incubated on ice for 15 minutes. Cells were then lysed byadding NP-40 to a final concentration of 0.67% and immediately vortexingfor 10 seconds.

The lysate was centrifuged at 20,000 g for 30 seconds at 4° C. to pelletnuclei. Nuclei were resuspended in buffer B (20 mM HEPES-K⁺, pH 7.9, 400mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF), incubated for 15minutes on ice with intermittent agitation and centrifuged at 20,000 gfor 5 minutes at 4° C. to prepare nuclear extracts.

In addition, a DNA probe (WT, SEQ ID NO: 30) corresponding to the mouseIL-31 promoter region containing the consensus SP1-binding sequence waslabelled with [γ-³²P] ATP (Perkin Elmer) using T4 polynucleotide kinase(Promega) and purified using illustra (trademark) MicroSpin (trademark)G-25 columns (GE Healthcare).

Protein-DNA binding was carried out as follows. 4 μg nuclear extractswere incubated in 9 μL of binding buffer (20 mM HEPES-K⁺, pH 7.9, 50 mMKCl, 3 mM MgCl₂, 10% glycerol, 1 mM DTT) supplemented with 2 μgpoly(dI-dC) (Sigma-Aldrich) in the presence or absence of unlabelledcompetitor DNA (WT (SEQ ID NO: 30) or its mutant (SEQ ID NO: 31)) for2.5 hours on ice, before addition of 1 μL of [³²P]-labelled probe (0.035pmol) and incubation at room temperature for 20 minutes.

Protein-DNA complexes were separated on a 6% nativepolyacrylamide/0.5×TBE gel at 4° C., dried onto a filter paper at 80° C.for 2 hours under vacuum and analyzed with the BAS2,000 BIO ImagingAnalyzer (Fuji Photo Film).

For supershift assays, 3 μg anti-SP1 antibody (Millipore) or 6 μganti-GFP antibody (Invitrogen) as a control were added to nuclearextracts and incubated on ice for 2.5 hours before addition ofradiolabelled probes.

(Chromatin Immunoprecipitation)

MEFs of wild type (WT) and EPAS1^(−/−) were cultured in 10 mL of DMEMmedium containing 10% FCS for 12 hours in two 100 mm culture dishes,followed by culturing in 1% 02 environment for 24 hours. The cells werethen cross-linked with 1% formaldehyde for 10 minutes at roomtemperature, and then glycine was added and reacted at room temperaturefor 5 minutes to neutralize. The cells were then washed with PBScontaining protease inhibitors, and then nuclei were extracted using acommercially available kit (model “Magna ChIP (trademark) HiSens kit”,Millipore) according to the manufacturer's protocol.

Isolated nuclei were resuspended in sonication buffer and sonicatedusing an ultrasound homogenizer (model “Cell disruptor 200”, Branson)with 16 sets of 10 pulses using the power set at “6” on ice for shearingchromatin DNA. Then, after centrifugation at 10,000 g for 10 minutes at4° C., sheared chromatin DNA was recovered and treated with RNase andproteinase K for quantifying DNA content.

The immunoprecipitation was carried out as follows. First, 3 to 7 μgchromatin DNA was mixed with magnetic Protein A/G beads preincubatedwith 2 μg anti-SP1 antibody (model “07-645”, Millipore) or 1 μg rabbitnormal IgG (Cell Signaling) and incubated at 4° C. overnight.

The magnetic beads were washed three times with buffer containingphysiologic salt and once with low salt buffer. Then, the magnetic beadswere treated with proteinase K at 65° C. for 2 hours, and heated at 95°C. for 15 minutes for inactivation of proteinase K and elution of boundchromatin DNA.

Eluted DNA was analyzed by quantitative real-time PCR using primers (SEQID NOs: 32 and 33) which amplify the mouse IL-31 promoter (−249/−97).

(Statistical Analysis)

Differences between groups were compared by unpaired one-tailedStudent's t test (in a case of two groups) or a one-way analysis ofvariance (in a case of multiple groups), followed by post hoc Bonferronitest. In addition, P values less than 0.05 were considered significant.

<I. Atopic Dermatitis in DOCK8^(−/−) AND Tg Mice>

Experimental Example 1

The AND is the TCR that recognizes a peptide (SEQ ID NO: 1) consistingof 88th to 103rd amino acids of Moth cytochrome c (MCC), which forms acomplex with MHC class II I-E^(K) molecule.

In order to investigate the influence of deficiency of DOCK8 on CD4⁺ Tcell differentiation and function, the present inventors crossed AND TCRtransgenic (Tg) mice with DOCK8^(−/−) mice. As a result, the presentinventors revealed that DOCK8^(−/−) AND Tg mice spontaneously developsevere atopic dermatitis by 14 to 15 weeks of age.

(a) of FIG. 1 is a representative photograph of 18-week-old DOCK8^(+/−)AND Tg mice. (b) of FIG. 1 is a representative photograph of 18-week-oldDOCK8^(−/−) AND Tg mice.

In addition, (c) of FIG. 1 is a graph showing the incidence of atopicdermatitis in male mice (n=20) having a genotype of DOCK8^(−/−) AND Tg.(d) of FIG. 1 is a graph showing the incidence of the atopic dermatitisin female mice (n=15) having the genotype of DOCK8^(−/−) AND Tg. In (c)and (d) of FIG. 1, the same number of DOCK8^(+/−) AND Tg littermateswere analyzed as controls.

As shown in (c) and (d) of FIG. 1, there was no significant differencein incidence between males and females. The atopic dermatitis developedat 7 to 8 weeks of age and deteriorated with increases of age. On theother hand, the same as DOCK8^(−/−) mice, the atopic dermatitis did notdevelop in DOCK8^(+/−) AND Tg mice.

Experimental Example 2

The scratching behavior of DOCK8^(−/−) AND Tg mice was analyzed. FIG. 2is a graph showing quantitative measurements of scratching behaviors per2 hours of DOCK8^(+/−) AND Tg mice and DOCK8^(−/−) AND Tg mice at 6weeks of age (n=8), 12 weeks of age (n=6), and 18 weeks of age (n=8).Values in the graph represent average value±standard deviation. Thesymbol “*” in the drawing indicates that there is a significantdifference at a risk ratio of less than 5%. As shown in FIG. 2,DOCK8^(−/−) AND Tg mice exhibited severe scratching behaviors at 12 and18 weeks of age. This result indicates that this dermatitis is veryitchy.

Experimental Example 3

Histological and immunohistochemical analysis of the skin of DOCK8^(−/−)AND Tg mice was performed. (a) and (b) of FIG. 3 are photographs of theresults of hematoxylin and eosin staining of the skin of the 18-week-oldDOCK8^(+/−) AND Tg mice ((a) of FIG. 3) and the DOCK8^(−/−) AND Tglittermate mice ((b) of FIG. 3). Scale bar indicates 50 μm.

(c) of FIG. 3 is a graph showing the total number of inflammatory cellsper 0.25 mm² in the skin of the DOCK8^(+/−) AND Tg mice (n=4 at eachweek of age) and the DOCK8^(−/−) AND Tg mice (n=4 at each week of age).Values in the graph represent average value±standard deviation. Thesymbol “*” in the drawing indicates that there is a significantdifference at a risk rate of less than 5%, and the symbol “**” indicatesthat there is a significant difference at a risk rate of less than 1%.

(a) and (b) of FIG. 4 are fluorescence microscopy photographs obtainedby staining the skin of the DOCK8^(+/−) AND Tg mice ((a) of FIG. 4) andthe DOCK8^(−/−) AND Tg mice ((b) of FIG. 4) with anti-CD3 antibody andanti-CD4 antibody. Scale bar indicates 50 μm. As a result, it wasrevealed that CD4⁺ T cells infiltrated into the skin of DOCK8^(−/−) ANDTg mice.

Subsequently, infiltration of CD8+ T cells and B cells was examined. (c)and (d) of FIG. 4 are fluorescence microscopy photographs obtained bystaining the skin of the DOCK8^(+/−) AND Tg mice ((c) of FIG. 4) and theDOCK8^(−/−) AND Tg mice ((d) of FIG. 4) with anti-CD3 antibody, anti-CD8antibody, and anti-B220 antibody. Scale bar indicates 50 μm. As aresult, it was recognized that there was no infiltration of CD8+ T cellsand B cells on the skin of DOCK8^(−/−) AND Tg mice.

Based on the above results, it became clear that, in the skin ofDOCK8^(−/−) AND Tg mouse, epidermal hyperplasia accompanied with massiveinfiltration of CD4⁺ T cells and hyperkeratosis were recognized, as inthe skin of patients with atopic dermatitis.

Experimental Example 4

The concentrations of IgE and IgG2b in the sera of DOCK8^(+/−) AND Tgmice and DOCK8^(−/−) AND Tg mice were measured. (a) and (b) of FIG. 5are graphs showing the concentrations of IgE ((a) of FIG. 5) and IgG2b((b) of FIG. 5) in sera of the DOCK8^(+/−) AND Tg mice and theDOCK8^(−/−) AND Tg mice at 6 weeks of age (n=13), 12 weeks of age (n=8and 9), and 18 weeks of age (n=4). The line in the graph indicates theaverage value, and the symbol “*” indicates that there is a significantdifference at a risk ratio of less than 5%.

As a result, the concentrations of IgE in the serum increased inDOCK8^(−/−) AND Tg mice at 12 weeks of age and 18 weeks of age, but theconcentration of IgG2b in the serum did not increase.

Experimental Example 5

The concentrations of IL-31 in the sera of DOCK8^(+/−) AND Tg mice andDOCK8^(−/−) AND Tg mice were measured. FIG. 6 is a graph showing aconcentration of IL-31 in the sera of the DOCK8^(+/−) AND Tg mice andthe DOCK8^(−/−) AND Tg mice at 6 weeks of age (n=6) and 18 weeks of age(n=6). The line in the graph indicates the average value, and the symbol“**” indicates that there is a significant difference at a risk ratio ofless than 1%.

As a result, the concentrations of IL-31 in the sera were significantlyincreased in 18-week-old DOCK8^(−/−) AND Tg mice, compared toDOCK8^(+/−) AND Tg mice (147.2±29.3 μg/mL vs. 41.5±28.6 μg/mL).

<II. DOCK8 Negatively Regulates IL-31 Production in CD4⁺ T Cells>

Experimental Example 6

The IL-31 is produced mainly from activated CD4⁺ T cells. The presentinventors compared the immunological function of CD4⁺ T cells ofDOCK8^(+/−) AND Tg mice and DOCK8^(−/−) AND Tg mice.

FIG. 7 is the results of flow cytometry analysis for thymocytes andperipheral lymph node cells of DOCK8^(+/−) AND Tg mice and DOCK8^(−/−)AND Tg mice of 6 to 8 weeks of age. In addition, FIG. 8 is the resultsof flow cytometry analysis for spleen cells of DOCK8^(+/−) AND Tg miceand DOCK8^(−/−) AND Tg mice of 6 to 8 weeks of age.

As a result, it was revealed that development of T cells in the thymusis normal even when DOCK8 is deficient. However, as reported inDOCK8^(−/−) mice, the proportion of T cells in peripheral lymph nodesand spleen was decreased in DOCK8^(−/−) AND Tg mice. In addition,regardless of the DOCK8 expression, peripheral T cells were CD4⁺ T cellsexpressing Vα11⁺Vβ3⁺ AND TCR.

Furthermore, the expression of CD44, which is an activation/memorymarker in CD4⁺ T cells, was similar in DOCK8^(+/−) AND Tg andDOCK8^(−/−) AND Tg mice at 6 to 8 weeks of age.

Experimental Example 7

The antigen-specific proliferation of CD4⁺ T cells from DOCK8^(+/−) ANDTg mice and DOCK8^(−/−) AND Tg mice was investigated. FIG. 9 is a graphshowing proliferation of CD4⁺ T cells from DOCK8^(+/−) AND Tg mice andDOCK8^(−/−) AND Tg mice when they are stimulated with MCC peptide (SEQID NO: 1) in the presence of spleen cells of B10.BR mice expressingI-E^(K).

As a result, as shown in FIG. 9, it was revealed that theantigen-specific proliferation of CD4⁺ T cells from DOCK8^(+/−) AND Tgmice and DOCK8^(−/−) AND Tg mice was in the comparable level.

Experimental Example 8

CD4⁺ T cells from DOCK8^(+/−) AND Tg mice and DOCK8^(−/−) AND Tg micewere primarily stimulated with MCC peptide and expression of cytokinegenes was examined.

(a) of FIG. 10 is a graph showing the expression of the IL-31 gene, (b)of FIG. 10 is a graph showing the expression of the IL-4 gene, and (c)of FIG. 10 is a graph showing the expression of IL-2 gene. In (a) to (c)of FIG. 10, the expression level of each gene was expressed as arelative value by setting the result of CD4⁺ T cells from DOCK8^(+/−)AND Tg mice as 1. In addition, the graph shows the averagevalue±standard deviation of four independent experimental results. Thesymbol “*” in the drawing indicates that there is a significantdifference at a risk ratio of less than 5%.

As a result, CD4⁺ T cells from DOCK8^(−/−) AND Tg mice showed increasedIL-31 transcripts at 24 hours after antigenic stimulation compared toCD4⁺ T cells derived from DOCK8^(+/−) AND Tg mice. On the other hand,expression of the IL-2 gene was equivalent in CD4⁺ T cells derived fromDOCK8^(+/−) AND Tg mice and CD4⁺ T cells derived from DOCK8^(−/−) AND Tgmice.

FIG. 11 is a graph showing the expression level of IL-31 gene in CD4⁺ Tcells from DOCK8^(+/−) AND Tg mice and from DOCK8^(−/−) AND Tg mice,after the secondary stimulation with anti-CDR antibody and anti-CD28antibody. Values in the drawing are relative values by setting theexpression level of the IL-31 gene in CD4⁺ T cells from DOCK8^(+/−) ANDTg mice without secondary stimulation as 1. The graph shows the averagevalue±standard deviation of three independent experimental results. Thesymbol “*” in the drawing indicates that there is a significantdifference at a risk rate of less than 5%, and the symbol “**” indicatesthat there is a significant difference at a risk rate of less than 1%.

As shown in FIG. 11, the above effect due to DOCK8 deficiency becamemore prominent when activated CD4⁺ T cells were recovered 96 hours afterantigen stimulation and re-stimulated with anti-CDR antibody.

The transcript of IL-31 reached 2860 times 3 hours after the secondarystimulation of CD4⁺ T cells from DOCK8^(−/−) AND Tg mice, compared tothe control sample without antigenic stimulation. This value was 27.3times when the amount of IL-31 transcript in CD4⁺ T cells fromDOCK8^(+/−) AND Tg mice were similarly analyzed.

Experimental Example 9

Expression level of IL-31 protein after secondary stimulation of CD4⁺ Tcells from DOCK8^(+/−) AND Tg mice and DOCK8^(−/−) AND Tg mice wasmeasured by ELISA method.

FIG. 12 is a graph showing measurement results. The graph shows averagevalue±standard deviation in representative results of three independentexperiments measured in 3 wells. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%.

As a result, in agreement with the results of Experimental Example 8, itwas revealed that CD4⁺ T cells from DOCK8^(−/−) AND Tg mice producelarge amounts of IL-31 upon antigen stimulation.

Experimental Example 10

As shown in (b) of FIG. 10, CD4⁺ T cells from the activated DOCK8^(−/−)AND Tg mice increased the expression of IL-4 gene.

Then, the present inventors investigated the influence of IL-4 on theexpression of the IL-31 gene in CD4⁺ T cells from DOCK8^(+/−) AND Tgmice and DOCK8^(−/−) AND Tg mice by treating them with anti-IL-4antibody in primary stimulation. For this purpose, anti-IL-4 antibodywas added to the medium during the primary stimulation of CD4⁺ T cellswith MCC peptide.

FIG. 13 is a graph showing the expression level of the IL-31 gene.Values in the graph are relative values by setting the expression levelof the IL-31 gene in CD4⁺ T cells from DOCK8^(+/−) AND Tg mice withoutsecondary stimulation as 1. The graph shows the average value±standarddeviation of three independent experimental results. The symbol “**” inthe drawing indicates that there is a significant difference at a riskratio of less than 1%.

As a result, anti-IL-4 antibody treatment reduced the expression levelof the IL-31 gene in both CD4⁺ T cells from DOCK8^(+/−) AND Tg mice andDOCK8^(−/−) AND Tg mice. However, the expression level of the IL-31 genein CD4⁺ T cells from DOCK8^(−/−) AND Tg mice was significantly higherthan CD4⁺ T cells from DOCK8^(+/−) AND Tg mice.

Based on the above result, it was revealed that DOCK8 negativelyregulates IL-31 production by CD4⁺ T cells independently of IL-4mediated signaling.

Experimental Example 11

In order to investigate whether the above findings can be extended toTCR with different antigen-specificity, the present inventors preparedand analyzed DOCK8^(−/−) mice expressing OTII TCR which recognize theovalbumin (OVA) peptide (SEQ ID NO: 4) presented by the I-A^(b)molecule.

FIG. 14 is the results of flow cytometry analysis for peripheral lymphnode cells of DOCK8^(+/−) OTII Tg mice and DOCK8^(−/−) OTII Tg mice of 6to 8 weeks of age.

As a result, it was revealed that CD4⁺ T cells develop normally inDOCK8^(−/−) OTII Tg mice as in DOCK8^(−/−) AND Tg mice.

Experimental Example 12

Subsequently, the antigen-specific proliferation of CD4⁺ T cells fromDOCK8^(+/−) OTII Tg mice and DOCK8^(−/−) OTII Tg mice was investigated.FIG. 15 is a graph showing proliferation of CD4⁺ T cells fromDOCK8^(+/−) OTII Tg mice and DOCK8^(−/−) OTII Tg mice when they arestimulated with OVA peptide (SEQ ID NO: 4) in the presence of spleencells of C57BL/6 mice expressing I-A^(b).

As a result, as shown in FIG. 15, it was revealed that theantigen-specific proliferation of CD4⁺ T cells from DOCK8^(+/−) OTII Tgmice and DOCK8^(−/−) OTII Tg mice was in the comparable level.

Experimental Example 13

Subsequently, CD4⁺ T cells from DOCK8^(+/−) OTII Tg mice and CD4⁺ Tcells derived from DOCK8^(−/−) OTII Tg mice were secondary stimulatedand the expression level of the IL-31 gene was measured. FIG. 16 is agraph of showing the expression of the IL-31 gene. The expression levelof the IL-31 gene was expressed as a relative value by setting theresult of CD4⁺ T cells from DOCK8^(+/−) OTII Tg mice as 1. The graphshows average value±standard deviation in representative results ofthree independent experiments. The symbol “*” in the drawing indicatesthat there is a significant difference at a risk ratio of less than 5%.

Experimental Example 14

Production of IL-31 protein after secondary stimulation of CD4⁺ T cellsfrom DOCK8^(+/−) OTII Tg mice and DOCK8^(−/−) OTII Tg mice was measuredby ELISA method.

FIG. 17 is a graph showing measurement results. The graph shows averagevalue±standard deviation in representative results of three independentexperiments measured in 2 wells. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%.

As shown in FIG. 16 and FIG. 17, it was revealed that CD4⁺ T cells fromDOCK8^(−/−) OTII Tg mice produce large amounts of IL-31 after secondarystimulation. These results indicate that DOCK8 generally acts as anegative regulator of IL-31 induction, regardless of the antigenspecificity of CD4⁺ T cells.

Experimental Example 15

Subsequently, the concentrations of IL-31 in sera of 18-week-oldDOCK8^(+/−) OTII Tg mice (n=7) and DOCK8^(−/−) OTII Tg mice (n=7) werecompared.

FIG. 18 is a graph showing the concentration of IL-31 in sera ofDOCK8^(+/−) OTII Tg mice and DOCK8^(−/−) OTII Tg mice. The lines in thegraph indicate average values.

As a result, it became clear that, unlike DOCK8^(−/−) AND Tg mice,DOCK8^(−/−) OTII Tg mice showed no increase in IL-31 concentration inthe serum.

Experimental Example 16

Subsequently, histological analysis of the skin of 18-week-oldDOCK8^(+/−) OTII Tg mice (n=4) and DOCK8^(−/−) OTII Tg littermate mice(n=4) was performed.

(a) and (b) of FIG. 19 are photographs of the results of hematoxylin andeosin staining of the skin of 18-week-old DOCK8^(+/−) OTII Tg mice ((a)of FIG. 19) and DOCK8^(−/−) OTII Tg littermate mice ((b) of FIG. 19).Scale bar indicates 50 μm.

(c) of FIG. 19 is a graph showing the total number of inflammatory cellsper 0.25 mm² in the skin of the DOCK8^(+/−) OTII Tg mice (n=4) and theDOCK8^(−/−) OTII Tg mice (n=4). Values in the graph represent averagevalue±standard deviation.

As a result, it became clear that, unlike DOCK8^(−/−) AND Tg mice,DOCK8^(−/−) OTII Tg mice did not develop atopic dermatitis.

These results suggest that CD4⁺ T cells of DOCK8^(−/−) AND Tg mice areself-reactive T cells and are continuously stimulated by undefinedautoantigens presented to I-A^(b) molecules. That is, AND TCR is a TCRhaving self-reactivity.

<III. Identification of EPAS1 as Master Control Factor of IL-31>

Experimental Example 17

To clarify the mechanism of overexpression of IL-31 by CD4⁺ T cells ofDOCK8^(−/−) AND Tg mice, the present inventors performed microarrayanalysis. As a result, it was revealed that 856 genes were highlyexpressed in CD4⁺ T cells of DOCK8^(−/−) AND Tg mice as compared withthose in CD4⁺ T cells of control DOCK8^(+/−) AND Tg mice after antigenstimulation.

These genes contained 40 putative transcription factors, one of whichwas EPAS1. Therefore, the expression of the IL-31 gene was examined byoverexpressing EPAS1 in wild-type CD4⁺ T cells.

First, CD4⁺ T cells of C57BL/6 mice were stimulated with plate-boundanti-CD3ε antibody and anti-CD28 antibody. Subsequently, the retroviralvector pMX-EPAS 1-IRES-GFP and pMX-IRES-GFP (control) that encodes GFPwith or without EPAS1, respectively, were introduced into CD4⁺ T cells,and the expression of IL-31 gene expression was measured.

FIG. 20 is a graph showing the expression level of IL-31 gene. Thevalues in the graph are relative values by setting the expression levelof IL-31 gene in the control sample (pMX-IRES-GFP transfection group)as 1. The graph shows the average value±standard deviation (n=6) ofthree independent experimental results. The symbol “*” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 5%.

As a result, the expression of the IL-31 gene following TCR stimulationwas significantly increased in CD4⁺ T cells overexpressing EPAS1, ascompared with the control.

Experimental Example 18

The EPAS1 gene in CD4⁺ T cells from DOCK8^(−/−) AND Tg mice was knockeddown and the influence thereof on the expression of IL-31 geneexpression was examined.

After siRNA specific for EPAS1 or control siRNA was introduced into CD4⁺T cells from DOCK8^(−/−) AND Tg mice, they were stimulated with antigen,and then the expression level of IL-31 gene was measured. The knockdownefficacy was examined by RT-PCR.

(a) of FIG. 21 is a graph showing the expression level of IL-31 gene.The values in the graph are relative values by setting the expressionlevel of the IL-31 gene in cells without antigen stimulation as 1. Thegraph shows the average value±standard deviation of three independentexperimental results. The symbol “*” in the drawing indicates that thereis a significant difference at a risk ratio of less than 5%. (b) of FIG.21 is a graph of the results of the RT-PCR.

As a result, it was revealed that knocking down EPAS1 gene significantlysuppresses the expression of IL-31 gene in the CD4⁺ T cells fromDOCK8^(−/−) AND Tg mice.

Experimental Example 19

Subsequently, the effect of the deletion of EPAS1 gene on IL-31 geneexpression in CD4⁺ T cells from DOCK8^(−/−) AND Tg mice was examined.

Specifically, CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice lackingEPAS1 genes specifically in T cells were generated, and the expressionlevel of IL-31 gene in CD4⁺ T cells from these mice was measured.

FIG. 22 is a graph showing the expression level of IL-31 gene. Thevalues in the graph are relative values by setting the expression levelof the IL-31 gene in cells without antigen stimulation as 1. The graphshows average value±standard deviation of the representative threeindependent experiments. In each experiment, the assays were performedwith triplicate wells. The symbol “**” in the drawing indicates thatthere is a significant difference at a risk ratio of less than 1%.

As a result, deletion of the EPAS1 gene in CD4⁺ T cells yielded the sameresult as in the case where EPAS1 gene was knocked down. That is, it wasrevealed that the expression of IL-31 gene in CD4⁺ T cells fromDOCK8^(−/−) AND Tg mice was significantly suppressed by the deletion ofEPAS1 gene.

Experimental Example 20

The incidence of atopic dermatitis, scratching behavior, andconcentration of IL-31 in serum in CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−)AND Tg mice were examined.

FIG. 23 is a representative photograph of 14-week-old CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice and control littermate mice.

FIG. 24 is a graph showing quantitative measurements of scratchingbehaviors per 2 hours of the 14-week-old CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice and the control littermate mice(n=4). Values in the graph represent average value±standard deviation.The symbol “**” in the drawing indicates that there is a significantdifference at a risk ratio of less than 1%.

(a) of FIG. 25 shows photographs of the skin of the 14-week-old CD4-CreEPAS 1^(lox/lox)DOCK8^(−/−) AND Tg mice and the control littermate micestained with hematoxylin and eosin. Scale bar indicates 50 μm.

(b) of FIG. 25 is a graph showing the total number of inflammatory cellsper 0.25 mm² in the skin of the 14-week-old CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice (n=4) and the control littermatemice (n=4). Values in the graph represent average value±standarddeviation. The symbol “**” in the drawing indicates that there is asignificant difference at a risk ratio of less than 1%.

FIG. 26 is a graph showing the concentration of IL-31 in sera of the14-week-old CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice (n=4) andthe control littermate mice (n=4). The lines in the graph indicateaverage values. The symbol “*” in the drawing indicates that there is asignificant difference at a risk ratio of less than 1%.

In FIGS. 23 to 26, CD4-Cre⁻ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice orCD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−) AND Tg mice were used as controlmice.

Based on the above results, no increase in the concentration of IL-31 inthe serum was observed in all the CD4-Cre⁺ EPAS1^(lox/lox)DOCK8^(−/−)AND Tg mice tested, and neither scratching behavior nor atopicdermatitis development was evident in CD4-Cre⁺EPAS1^(lox/lox)DOCK8^(−/−)AND Tg mice.

Therefore, it was revealed that EPAS1 functions as a master regulator ofIL-31 induction in CD4⁺ T cells and is essential for the development ofatopic dermatitis in DOCK8^(−/−) AND Tg mice.

Experimental Example 21

Subsequently, how EPAS1 activates the promoter of IL-31 was examined.

The present inventors produced a reporter construct in which theluciferase gene was ligated downstream of the IL-31 promoter sequence(−1367 to −1). In addition, an expression vector of a mutant of EPAS1was produced.

(a) of FIG. 27 is a schematic diagram of EPAS1 mutants used in thepresent experimental example. Subsequently, the reporter construct wastransfected into mouse embryonic fibroblasts (MEF) with the geneencoding EPAS1 mutant and the activity of the IL-31 promoter wasmeasured.

(b) of FIG. 27 is a graph showing the activity of IL-31 promoter in thepresence of wild-type and mutant EPAS1. The value of the graph shows theaverage value±standard deviation of four independent experiments. Thesymbol “*” in the drawing indicates that there is a significantdifference at a risk rate of less than 5%, and the symbol “**” indicatesthat there is a significant difference at a risk rate of less than 1%.

As a result, it became clear that activation of IL-31 promoter wasinduced in the presence of wild-type EPAS1. However, activation of IL-31promoter was not induced by the mutant EPAS1 lacking the activationdomain at the N-terminal side (N-TAD) or the activation domain at theC-terminal side (C-TAD).

Experimental Example 22

Subsequently, the reporter construct of Experimental Example 21 and theexpression vector encoding wild-type EPAS1 with siRNA specific for Arylhydrocarbon receptor nuclear translocator (ARNT) gene or siRNA specificfor specificity protein 1 (SP1) gene were introduced into MEF, and theactivity of IL-31 promoter was measured.

(a) of FIG. 28 is a graph showing the activity of the IL-31 promoter.The value of the graph shows the average value±standard deviation ofthree independent experimental results. The symbol “*” in the drawingindicates that there is a significant difference at a risk rate of lessthan 5%, and the symbol “*” indicates that there is a significantdifference at a risk rate of less than 5%. (b) of FIG. 28 arephotographs of Western blotting showing the efficacy of knockdown ofARNT gene. (c) of FIG. 28 are photographs of Western blotting showingthe efficacy of knockdown of SP1 gene.

As a result, it was revealed that knockdown of the ARNT gene did notaffect EPAS1-mediated IL-31 promoter activation. On the other hand, itwas revealed that, EPAS1-mediated IL-31 promoter activation wasdecreased when the SP1 gene was knocked down.

The above results indicate that SP1, but not ARNT, is essential forEPAS1-mediated IL-31 promoter activation.

Experimental Example 23

Subsequently, the present inventors generated a reporter construct wherethe IL-31 promoter region used in Experimental Example 21 was deleted,and introduced it into MEF together with the expression vector encodingwild-type EPAS1 to measure the IL-31 promoter activation.

FIG. 29 is a graph showing the activity of IL-31 promoter. The value ofthe graph shows the average value±standard deviation of threeindependent experimental results. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%.

As a result, the present inventors identified a region important forEPAS1-mediated IL-31 promoter activation. This region contained “GCGC”at −118 to −115, which is consensus sequence of the SP1 binding site.

Experimental Example 24

Subsequently, the present inventors generated a reporter construct wherea site-specific mutation was introduced into the IL-31 promoter, andintroduced into MEF together with the expression vector encodingwild-type EPAS1 to measure IL-31 promoter activation.

FIG. 30 is a graph showing the activity of IL-31 promoter. The term“#1;CAA” in the drawing means that “TGG” positioned in the #1 region(−135 to −133) of the IL-31 promoter shown in the lower part of FIG. 30was mutated to “CAA”, and the same applies hereinafter. The value of thegraph shows the average value±standard deviation of three independentexperimental results. The symbol “**” in the drawing indicates thatthere is a significant difference at a risk ratio of less than 1%.

As a result, it became clear that mutation of the SP1 binding sequence“GCGC” located at −118 to −115 of the IL-31 promoter to “AAGT” resultsin nearly complete loss of EPAS1-mediated IL-31 promoter activation.This result shows that the SP1 binding sequence located at −118 to −115is important for the EPAS1-mediated IL-31 promoter activation.

Experimental Example 25

Subsequently, whether SP1 binds to the IL-31 promoter was investigatedby Electrophoretic mobility shift assay (EMSA).

(a) and (b) of FIG. 31 are photographs of the results of EMSA. In thedrawing, the term “C” indicates SP1-DNA complex and the term “SS”indicates super shift. The term “NE” means nuclear extract, the term“WT” means wild type sequence, and the term “Mut” means mutant typesequence.

As a result, it became clear that SP1 was definitely bound to the IL-31promoter. In addition, it was revealed that SP1 does not bind to a DNAfragment where “GCGC”, the SP1 binding sequence located at −118 to −115of the IL-31 promoter, was mutated to “AAGT”.

Experimental Example 26

Subsequently, the effect of EPAS1 on the binding of SP1 to the IL-31promoter was examined by chromatin immunoprecipitation (ChIP).

FIG. 32 is a graph of the results of ChIP assay. The value of the graphshows the average value±standard deviation of seven independentexperimental results. The symbol “*” in the drawing indicates that thereis a significant difference at a risk ratio of less than 5%.

As a result, it was revealed that the binding of SP1 to the IL-31promoter was markedly decreased in the absence of EPAS1.

Based on the result, it became clear that the EPAS1 activates the IL-31promoter independently of the ARNT, but in collaboration with the SP1.

The present inventors also carried out ChIP using CD4⁺ T cells insteadof MEF and examined the effect of EPAS1 on the binding of SP1 to theIL-31 promoter. As a result, even when CD4⁺ T cells were used, the sameresults were obtained as seen in the MEFs. That is, it was revealed thatthe binding of SP1 to the IL-31 promoter was markedly decreased in theabsence of EPAS1.

<IV. DOCK8 Functions as Adapter to Control Nuclear Translocation ofEPAS1>

Experimental Example 27

EPAS1 translocates from the cytoplasm to the nucleus and mediates IL-31promoter activation. Therefore, the present inventors examined theintracellular localization of EPAS1 among wild-type MEF and DOCK8^(−/−)MEF in order to elucidate the mechanism by which DOCK8 negativelycontrols IL-31 induction.

First, anti-EPAS1 antibody was verified by immunofluorescence staining.(a) of FIG. 33 shows fluorescence microscopy photographs of EPAS1^(−/−)MEF stained with anti-EPAS1 antibody. DAPI was used to stain thenucleus. Scale bar indicates 20 μm. (b) of FIG. 33 shows fluorescencemicroscopy photographs of wild-type MEF stained with the anti-EPAS1antibody. As a result, the validity of anti-EPAS1 antibody was checked.

Subsequently, the intracellular localization of EPAS1 in wild-type (WT)and DOCK8^(−/−) MEF under normal and hypoxia conditions was examined byimmunofluorescence staining. (a) and (b) of FIG. 34 are fluorescencemicroscopy photographs of the immunofluorescence staining. DAPI was usedto stain the nucleus. Scale bar indicates 20 μm. (c) of FIG. 34 is agraph showing the proportion of cells with nuclear localization ofEPAS1. The value of the graph shows the average value±standard deviationof four independent experiments. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%.

As a result, EPAS1 was translocated to the nucleus under hypoxia in bothwild-type and DOCK8^(−/−) MEFs. In addition, the nuclear localization ofEPAS1 in DOCK8^(−/−) MEF was remarkably increased under normalconditions as compared with that in wild-type MEF.

Experimental Example 28

Mutant DOCK8 (ΔN and ΔDHR2) and wild-type DOCK8 (WT) were expressed inDOCK8^(−/−) MEF and the subcellular localization of EPAS1 was examined.

(a) of FIG. 35 are fluorescence microscopy photographs showingintracellular localization of EPAS1 under normal and hypoxia conditionsby immunofluorescence staining. DAPI was used to stain the nucleus.Scale bar indicates 20 μm. (b) of FIG. 35 is a graph showing theproportion of cells with nuclear localization of EPAS1. The value of thegraph shows the average value±standard deviation of four independentexperimental results. The symbol “*” in the drawing indicates that thereis a significant difference at a risk rate of less than 5%, and thesymbol “**” indicates that there is a significant difference at a riskrate of less than 1%.

As a result, it was revealed that accumulation of EPAS1 in the nucleusdue to deletion of DOCK8 disappears by expressing wild-type DOCK8 inDOCK8^(−/−) MEF. Similar results were also obtained when the DOCK8mutant (ΔDHR2) lacking DOCK homology region (DHR)-2 domain essential forCdc42 activation was expressed in DOCK8^(−/−) MEF.

However, when the mutant (ΔN) lacking 527 amino acids at the N terminalof DOCK8 was expressed in DOCK8^(−/−) MEF, accumulation of EPAS1 in thenucleus could not be suppressed. This result shows that the N-terminalregion of DOCK8 is important for controlling intracellular localizationof EPAS1.

Experimental Example 29

By the way, mammalian STE 20-like kinase 1 (MST 1) is a serine/threoninekinase involved in T cell adhesion, migration, proliferation, andapoptosis. As a result of searching for binding proteins, the presentinventors revealed that DOCK8 binds to MST1 via N-terminal regionthereof.

(a) of FIG. 36 is the result of immunoprecipitation showing that DOCK8binds to MST1 via the N-terminal region.

Subsequently, DOCK8, MST1, and EPAS1 were co-expressed in 293T(HEK-293T) cells, human embryonic kidney cells, and immunoprecipitationwas performed. (b) of FIG. 36 are photographs showing the result ofimmunoprecipitation. As a result, it became clear that EPAS1 and MST1were co-immunoprecipitated irrespective of the presence of DOCK8.

This result suggests that DOCK8 binds to MST1, thereby regulating theintracellular localization of EPAS1.

Experimental Example 30

Subsequently, the effect of knockdown of MST1 gene on nuclearlocalization of EPAS1 in the nucleus was examined in wild-type MEF.

The shRNA of MST1 was introduced into wild-type MEF using pSUPERretro-puro sh MST1 retroviral vector, and MST1 was knocked down. FIG. 37are photographs of the results showing the efficacy of knockdown of MST1gene in Western blotting.

Subsequently, intracellular localization of EPAS1 under normal andhypxia conditions was analyzed by immunofluorescence staining forwild-type (WT) MEF, DOCK8^(−/−) MEF, and MEF where MST1 genes wereknocked down.

(a) to (c) of FIG. 38 are fluorescence microscopy photographs of theresults of the immunofluorescence staining. (a) of FIG. 38 shows resultsof wild-type MEF, (b) of FIG. 38 shows results of DOCK8^(−/−) MEF, and(c) of FIG. 38 shows results of MEF where the MST 1 gene was knockeddown. DAPI was used to stain the nucleus. Scale bar indicates 20 μm. (d)of FIG. 38 is a graph showing the proportion of cells with nuclearlocalization of EPAS1. The value of the graph shows the averagevalue±standard deviation of three independent experimental results. Thesymbol “**” in the drawing indicates that there is a significantdifference at a risk ratio of less than 1%.

As a result, it was revealed that knockdown of the MST1 gene markedlyincreases nuclear localization of EPAS1.

Experimental Example 31

The expression of the IL-31 gene was examined when MST1 gene was knockeddown in CD4⁺ T cells from DOCK8^(+/−) AND Tg mice.

Following transfection of siRNA specific for MST1 and control siRNA intoCD4⁺ T cells from DOCK8^(+/−) AND Tg mice, they were stimulated withantigen, and then the expression level of the IL-31 gene was measured.The knockdown efficacy was examined by RT-PCR.

(a) of FIG. 39 is a graph showing the expression level of IL-31 gene.The values in the graph are relative values by setting the expressionlevel of the IL-31 gene in cells without antigen stimulation as 1. Thegraph shows the average value±standard deviation (n=4) of twoindependent experimental results. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%. (b) of FIG. 39 is a graph of the results of the RT-PCR.

As a result, it was revealed that knocking down the MST1 genesignificantly increased the expression of the IL-31 gene in CD4⁺ T cellsfrom DOCK8^(+/−) AND Tg mice.

The above results indicate that the DOCK8-MST1 axis negatively regulatesthe induction of IL-31 by inhibiting the nuclear translocation of EPAS1.

<V. Role of EPAS1 in CD4⁺ T Cells of Patients with Atopic Dermatitis>

Experimental Example 32

Since it became clear that EPAS1 is essential for the induction of IL-31in murine CD4⁺ T cells, the present inventors examined the role of EPAS1in human CD4⁺ T cells.

The CD4⁺ T cells from the patients with atopic dermatitis and healthyvolunteers were cultured for 6 hours in a 24-well plate coated with orwithout anti-CD3ε antibody, and the expression of DOCK8 was examined byWestern blotting.

FIG. 40 shows photographs of the results of the Western blotting. As aresult, no significant difference in the expression of DOCK8 between thepatients with atopic dermatitis and healthy people (controls) wasobserved.

Experimental Example 33

Subsequently, the concentrations of IL-31 in sera of the patients withatopic dermatitis and healthy people (controls) were measured forcomparison. FIG. 41 is a graph showing the concentration of IL-31 inserum (n=6). The lines in the graph indicate average values. The symbol“**” in the drawing indicates that there is a significant difference ata risk ratio of less than 1%.

As a result, it was revealed that the concentration of IL-31 in theserum of patients with atopic dermatitis was remarkably increased ascompared with that of healthy controls.

Experimental Example 34

Subsequently, expression of cytokine genes in CD4⁺ T cells following TCRstimulation was examined.

CD4⁺ T cells from the patients with atopic dermatitis and healthycontrols were stimulated with anti-CD3ε antibody, and expression levelsof IL-31 gene and IL-2 gene were measured. (a) of FIG. 42 is a graphshowing the expression level of the IL-31 gene. (b) of FIG. 42 is agraph showing the expression level of the IL-2 gene.

The values of the graph are relative values by setting the expressionlevel of each cytokine gene in CD4⁺ T cells from the healthy controlswithout TCR stimulation as 1; and the data show average value±standarddeviation (n=6). The symbol “*” in the drawing indicates that there is asignificant difference at a risk ratio of less than 5%.

As a result, as seen in the CD4⁺ T cells from DOCK8^(−/−) AND Tg mice,the expression level of IL-31 gene following TCR stimulation wasremarkably high in the CD4⁺ T cells from the patients with atopicdermatitis, compared to that in CD4⁺ T cells from healthy controls, butthe expression levels of the IL-2 gene were comparable.

Experimental Example 35

Subsequently, the present inventors examined the effect of EPAS1inhibitor on IL-31 induction in CD4⁺ T cells from the patients withatopic dermatitis. For this purpose, EPAS1 inhibitor, FM19G11 andHIFVII, were used. Although the precise mechanism has not beenelucidated, FM19G11 is known to inhibit the expression of EPAS1.

First, the validity of anti-EPAS1 antibody was confirmed by Westernblotting. Following expression of pcDNA-EPAS1-FLAG encoding FLAG-taggedhuman EPAS1 in HEK-293T cells, cell lysates were analyzed by the Westernblotting using anti-FLAG antibody and anti-EPAS1 antibody. FIG. 43 showsphotographs of the results of the Western blotting. As a result, thevalidity of anti-EPAS1 antibody was checked.

Subsequently, the influence of the EPAS1 inhibitor on the expression ofEPAS1 in the CD4⁺ T cells from the patients with atopic dermatitis andhealthy controls was examined.

FIG. 44 shows photographs of the results of the Western blotting. Thephotographs show representative results of three independentexperiments. As a result, it was confirmed that FM19G11 markedlydecreased the expression level of EPAS1 protein in human CD4⁺ T cells.

Experimental Example 36

Subsequently, the influence of EPAS1 inhibitors, FM19G11 (n=6) andHIFVII (n=3), on the cytokine gene expression in CD4⁺ T cells from thepatients with atopic dermatitis was examined following TCR stimulation.

(a) of HG 45 is a graph showing the effect of FM19G11 on the expressionlevel of IL-31 gene. (b) of FIG. 45 is a graph showing the effect ofFM19G11 on the expression level of the IL-2 gene. (c) of FIG. 45 is agraph showing the effect of HIFVII on the expression level of the IL-31gene. In (a) to (c) of FIG. 45, the data in the graph are relativevalues by setting the expression level of each cytokine in CD4⁺ T cellsfrom the patients without TCR stimulation as 1. The graph shows theaverage value±standard deviation. The symbol “**” in the drawingindicates that there is a significant difference at a risk ratio of lessthan 1%.

As a result, when CD4⁺ T cells from the patients with atopic dermatitiswere treated with FM19G11, the expression of the IL-31 gene followingTCR stimulation was markedly suppressed, but the expression levels ofthe IL-2 gene were comparable. On the other hand, when CD4⁺ T cells fromthe patients with atopic dermatitis were treated with HIFVII, theinhibitory effect on IL-31 gene expression was not observed.

Experimental Example 37

It is known that HIFVII binds to the PAS-B domain of EPAS1 and inhibitsthe binding between EPAS1 and ARNT. In order to confirm the abovedescription, ARNT and EPAS1 were co-expressed in HEK-293T cells, andimmunoprecipitation was performed in the presence or absence of HIFVII.(a) and (b) of HG 46 are photographs showing the result ofimmunoprecipitation. As a result, it was confirmed that the HIFVIItreatment inhibited binding of ARNT and EPAS1.

These results indicate that in the CD4⁺ T cells from the patients withatopic dermatitis, EPAS1 regulates the induction of IL-31 geneirrespective of binding to ARNT.

<VI. Atopic Dermatitis in DOCK8 Conditional Knockout Mice>

Experimental Example 38

Whether conditional knockout mice where DOCK8 was specifically absentfrom CD4⁺ T cells expressing AND TCR developed atopic dermatitis wasinvestigated.

First, DOCK8^(lox/lox) mice were produced by a standard method.Subsequently, DOCK8^(lox/lox) mice were crossed with AND TCR Tg mice toobtain DOCK8^(lox/lox) AND Tg mice. In addition, DOCK8^(lox/lox) AND Tgmice were crossed with CD4-Cre Tg mice to obtain CD4-Cre⁺DOCK8^(lox/lox)AND Tg mice.

DOCK8 was specifically absent from CD4⁺ T cells inCD4-Cre⁺DOCK8^(lox/lox) AND Tg mice. CD4-Cre⁺DOCK8^(lox/lox) AND Tg micewere maintained under specific-pathogen-free (SPF) conditions and theincidence of dermatitis was examined. Age- and sex-matched littermateswere used as controls.

The state of dermatitis was evaluated according to the Severity Scoringof Atopic Dermatitis (SCORAD) index. Specifically, four items of (1)erythema/hemorrhage, (2) scaling/dryness, (3) edema, and (4)excoriation/erosion were scored as 0: absence, 1: mild, 2: moderate, and3: severe with scores of total 12 points were evaluated.

FIG. 47 is a graph showing the change in the SCORAD index of DOCK8conditional knockout mice (KO, n=4) and control mice (heterozygosity,n=4). The values in the graph represent average value±standarddeviation.

As a result, it became clear that the DOCK8 conditional knockout micedeveloped atopic dermatitis after 7 weeks of age, whereas the controlmice did not develop dermatitis.

This result further supports that the gene mutation by whichtri-molecular complex comprising DOCK8 protein, MST1 protein, and EPAS1protein is not formed in the CD4⁺ T cells causes development of atopicdermatitis.

<VII. Investigation of CD4⁺ T Cells Derived from DOCK8^(−/−) OTII TgMice>

Experimental Example 39

In Experimental Example 16, it is indicated that DOCK8^(−/−) OTII Tgmice do not develop atopic dermatitis. Therefore, CD4⁺ T cells fromDOCK8^(−/−) OTII Tg mice were adoptively transferred into CAG-OVA miceexpressing the OVA antigen systemically, and whether itching was inducedwas investigated.

Specifically, first, CD4⁺ T cells from DOCK8^(+/−) OTII Tg mice orlittermate DOCK8^(−/−) OTII Tg mice were cultured in the presence of OVA323-339 peptide (SEQ ID NO: 4, 1 μg/mL) with T cell-depleted, irradiatedspleen cells (5×10⁶ cells/well). Subsequently, activated CD4⁺ T cellfrom each mice were intravenously administered to CAG-OVA mice at a doseof 5.7×10⁶ cells/mouse. Subsequently, observation of the scratchingbehaviors started 5 hours after the administration of the cells.

FIG. 48 is a graph showing quantitative measurements of scratchingbehaviors per 2 hours of CAG-OVA mice transferred with CD4⁺ T cells fromDOCK8^(+/−) OTII Tg mice or with CD4⁺ T cells from the DOCK8^(−/−) OTIITg mice (n=4 for each group). Values in the graph represent averagevalue±standard deviation. The symbol “*” in the drawing indicates thatthere is a significant difference at a risk ratio of less than 5%.

As a result, as shown in FIG. 48, it was revealed that the CAG-OVA micetransferred with CD4⁺ T cells from DOCK8^(−/−) OTII Tg mice exhibitsevere scratching behavior. This result indicates that itching can beinduced by transferring CD4⁺ T cells from DOCK8^(−/−) OTII Tg mice intothe CAG-OVA mice.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to clarify themechanism regulating IL-31 production and provide an atopic dermatitismodel non-human animal.

1.-26. (canceled)
 27. A method for screening a therapeutic agent foratopic dermatitis, comprising: expressing EPAS1 gene in a cell, intowhich a reporter construct in which a reporter gene is linked downstreamof an IL-31 promoter is introduced in the presence of a test substanceto quantitatively determine an expression level of the reporter gene,wherein a decrease of the expression level of the reporter gene whencompared to the expression level of the reporter gene in the absence ofthe test substance indicates that the test substance is the therapeuticagent for atopic dermatitis.
 28. A method for screening a therapeuticagent for atopic dermatitis, comprising: expressing EPAS1 gene in Tcells, and stimulating TCR of the T cells with anti-CD3ε antibody in thepresence of a test substance to quantitatively determine an expressionlevel of IL-31, wherein a decrease of the expression level of IL-31 whencompared to the expression level of IL-31 in the absence of the testsubstance indicates that the test substance is the therapeutic agent foratopic dermatitis.
 29. A method for screening a therapeutic agent foratopic dermatitis, comprising: stimulating TCR of DOCK8^(−/−) T cells orMST1^(−/−) T cells, into which a reporter construct in which a reportergene is linked downstream of an IL-31 promoter is introduced in thepresence of a test substance to quantitatively determine an expressionlevel of the reporter gene, wherein a decrease of the expression levelof the reporter gene when compared to the expression level of thereporter gene in the absence of the test substance indicates that thetest substance is the therapeutic agent for atopic dermatitis.
 30. Themethod for screening a therapeutic agent for atopic dermatitis accordingto claim 29, wherein the DOCK8^(−/−) T cells is DOCK8^(−/−) CD4⁺ Tcells, and the MST1^(−/−) T cells is MST1^(−/−) CD4⁺ T cells.
 31. Themethod for screening a therapeutic agent for atopic dermatitis accordingto claim 30, wherein the DOCK8^(−/−) CD4⁺ T cells is DOCK8^(−/−) AND TgCD4⁺ T cells or DOCK8^(−/−) OTII Tg CD4⁺ T cells, and the MST1^(−/−)CD4⁺ T cells is MST1^(−/−) AND Tg CD4⁺ T cells or MST1^(−/−) OTII TgCD4⁺ T cells.
 32. A method for screening a therapeutic agent for atopicdermatitis, comprising: expressing EPAS1 gene in DOCK8^(−/−) cells or inMST1^(−/−) cells in the presence of a test substance to quantitativelydetermine the level of nuclear EPAS1 protein, wherein a decrease of thelevel of nuclear EPAS1 protein when compared to the level of nuclearEPAS1 protein in the absence of the test substance indicates that thetest substance is the therapeutic agent for atopic dermatitis.